Transgenic plants presenting a modified inulin producing profile

ABSTRACT

A method is disclosed for producing a transgenic plant with a modified inulin producing profile comprising in its genome a combination of one or more expressible 1-SST enzyme encoding genes and one or more expressible 1-FFT enzyme encoding genes, wherein either of these genes or both of them comprise one or more recombinant genes containing one or more 1-SST, respectively 1-FFT, enzyme encoding DNA sequences of plant origin or an expressible homologous sequence thereof. The invention also relates to a method for modifying and controlling the inulin profile of plants and to a method for producing inulin from said transgenic plants. Furthermore, a novel cDNA sequence of a 1-SST enzyme encoding gene of  Helianthus tuberosus  and a novel cDNA sequence of a 1-FFT enzyme encoding gene of  Cichorium intybus  are disclosed, novel recombinant DNA constructs and genes derived thereof, as well as novel combinations of expressible 1-SST and 1-FFT enzyme encoding genes. Moreover, the invention also relates to novel polypeptides, homologues thereof and fragments thereof, which have 1-SST activity of 1-FFT activity, and to antibodies capable of specifically binding one or more of them.

FIELD OF THE INVENTION

The present invention relates to transgenic plants presenting a modifiedinulin producing profile, to a method for producing said plants, to amethod for modifying and controlling the inulin producing profile ofplants and to a method for producing inulin from said transgenic plants.

Furthermore, the present invention relates to novel 1-SST and 1-FFTenzyme encoding DNA sequences, to novel recombinant DNA constructs andrecombinant genes derived thereof, to novel combinations of expressible1-SST and 1-FFT enzyme encoding genes, as well as to novel polypeptidesor fragments thereof presenting 1-SST and/or 1-FFT activity, and toantibodies capable of binding to them.

BACKGROUND AND PRIOR ART

Inulin is a fructan type carbohydrate polymer which occurs as apolydisperse composition in many plants and can also be produced bycertain bacteria and fungi. Inulin from plant origin consists of apolydisperse composition of mainly linear chains composed of fructoseunits, mostly terminating in one glucose unit, which are linked to eachother through β(2-1) fructosyl-fructose linkages.

Inulin can be generally represented, depending from the terminalcarbohydrate unit, by the formulae GF_(n) and F_(m), wherein G and Frespectively represent a glucose unit and a fructose unit, n is aninteger representing the number of fructose units linked to the terminalglucose unit, and m is an integer representing the number of fructoseunits linked to each other in the polyfructose chain.

The number of saccharide units (fructose and glucose units) in onemolecule, i.e. the values n+1 and m in the above formulae, are commonlyreferred to as the degree of polymerisation, represented by DP. Oftenalso the parameter (number) average degree of polymerisation,represented by ({overscore (DP)}) is used, which is the value {overscore(DP)}_(n) calculated, after complete hydrolysis and considering that innative inulins the F_(m) fraction is negligible, as follows:${\overset{\_}{DP}}_{n} = {\frac{{total}\quad \% \quad F}{{total}\quad \% \quad G} + 1}$

In the equation % refers to weight percent (wt %). Furthermore, in thiscalculation the saccharides glucose (G), fructose (F) and saccharose(GF) which are present in the polydisperse polysaccharide, should not betaken into account. The average degree of polymerisation is thus the({overscore (DP)}_(n)) of inulin, herein interchangeably referred to inshort as ({overscore (DP)}) inulin or ({overscore (DP)}) (De Leenheer,1996).

The polysaccharide chains of native inulin from plant origin generallyhave a degree of polymerisation (DP) ranging from 3 to about 100,whereas the ({overscore (DP)}) of the native inulin largely depends fromthe plant source, the growth phase of the plant, the harvesting time andthe storage conditions. The ({overscore (DP)}) of isolated inulinlargely depends on the ({overscore (DP)}) of the native inulin and onthe process conditions used for the extraction, purification andisolation of the inulin from the plant or plant parts.

By native inulin or crude inulin is meant herein inulin that has beenextracted from plants or parts of plants, without applying any processto increase or decrease the ({overscore (DP)}), while taking precautionsto inhibit the plant's own hydrolase activity and to avoid hydrolysis.The ({overscore (DP)}) of native inulin thus essentially corresponds tothe ({overscore (DP)}) of the inulin as present in the plant or plantparts.

The isolated inulin obtained from plants or plant parts throughconventional manufacturing techniques, commonly including extraction,purification and isolation, without any process to modify the({overscore (DP)}) of the native inulin, is termed herein,interchangeably, standard ({overscore (DP)}) grade inulin or standardgrade inulin. As a consequence of the manufacturing process, the({overscore (DP)}) of standard grade inulin is usually about 1 to 1.5lower than the ({overscore (DP)}) of the native inulin.

Inulin molecules with a DP≦10 are commonly termed oligofructose,inulo-oligosaccharides or fructo-oligosaccharides (in short FOS). Both,inulin chains with a DP≦10 and inulin chains with a DP>10, are embracedherein by the term inulin.

By inulin profile is meant the relative composition of the polydisperseinulin as formed by the individual components including glucose,fructose, sucrose and individual inulin chains, including thedistribution pattern of the polyfructose (inulin) chains.

Linear inulin is common in a specific plant family, the Asteraceae,including the plant species Jerusalem artichoke (Helianthus tuberosus)and chicory (Cichorium intybus). Inulin is commonly stored in tap roots(chicory) or in tubers (Jerusalem artichoke) and acts as a storagereserve for regrowth of the sprout after the winter period.

Accordingly, typical sources for the production of inulin at industrialscale are roots of chicory and, on a much smaller scale, tubers ofJerusalem artichoke, in which inulin can be present in concentrations ofabout 14% to 18% by weight on fresh weight. Inulin can be readilyextracted from these plant parts, purified and optionally fractionatedin order to remove impurities, monosaccharides, disaccharides andundesired oligosaccharides, as for example described in PCT patentapplication WO 96/01849.

Conventional processing of roots of chicory yields a standard gradeinulin, containing about 8 wt % of mono- and di-saccharides (includingglucose, fructose and sucrose) and a polydisperse mixture of inulinmolecules with a DP ranging from 3 to about 60 and a ({overscore (DP)})of about 10. The DP of the inulin molecules of standard grade inulinfrom Jerusalem Artichoke tubers ranges from 3 to about 40 whereas the({overscore (DP)}) is about 7.

It is known that in Asteraceaous plants, including Jerusalem artichokeand chicory, inulin molecules are synthesised by the concerted action oftwo enzymes:sucrose:sucrose 1-fructosyltransferase (in short 1-SSTenzyme or 1-SST, used interchangeably) and fructan:fructan1-fructosyltransferase (in short 1-FFT enzyme or 1-FFT, usedinterchangeably) (Koops and Jonker, 1994 and 1996). Both 1-SST and 1-FFTare active during the period of inulin synthesis and accumulation:

1-SST catalyses the initial reaction of inulin biosynthesis, theconversion of sucrose into the smallest inulin molecule, thetrisaccharide kestose (GFF), according to:

GF+GF→GFF+G  (1)

1-FFT catalyses the redistribution of terminal fructosyl units (−F)between inulin molecules, which results in a stepwise increase in chainlength, according to:

GFF_(n)+GFF_(m)→GFF_(n−1)+GFF_(m+)1,  (2)

(wherein n and m are integers >0)

Some examples of this type of reaction are

GFF+GFF→GFFF+GF  (2a)

GFFF+GFFF→GFFFF+GFF  (2b)

GFFFF+GFFFF→GFFFFF+GFFF  (2c)

An essential difference between the 1-SST enzyme and the 1-FFT enzyme isthat the 1-FFT enzyme cannot catalyse reaction (1). In contrast, the1-SST enzyme, next to reaction (1), can catalyse reactions of type (2)yielding inulin molecules with a low DP (catalysis by known 1-SSTenzymes being able to yield inulin molecules with a DP up to about 5).

Accordingly, in plants both the 1-SST and the 1-FFT enzymes arecontributing to inulin synthesis and the profile of native inulin isdetermined, inter alia, by sucrose supply, expression of the 1-SSTenzyme encoding genes and 1-FFT enzyme encoding genes and the kineticproperties and relative activity of the 1-SST and 1-FFT enzymes whichmay be controlled by the relative expression of the 1-SST and the 1-FFTenzyme encoding genes.

Inulin is an edible, water soluble polydisperse polysaccharidecomposition which is used in the manufacture of many food and feedproducts, drinks and non-food products. In food, feed and drinks, inulincan be used, inter alia, as a bulking agent as well as a total orpartial substitute for sugar and/or fat. Furthermore, inulin can beadded to food, feed and drinks to enrich them with soluble fibres havingprebiotic properties. Moreover, inulin can also be used as a componentof prophylactic and therapeutic compositions. Besides, inulin with a({overscore (DP)}) of about 10 and more is commonly used at industrialscale as starting material for the manufacture of oligofructose and offructose, which both are increasingly used in industry as sweeteners,particularly in drinks and fruit compositions.

Usually different applications require inulin with a different profile.For example for use as (oligofructose) sweetener, the inulin moleculesshould have a low DP, preferably about 3 to 8, whereas for use as fatreplacer inulin should preferably have a ({overscore (DP)}) higher than15. For non-food applications inulin may have to be derivatised, forexample to obtain carboxymethylated inulin which can be used assequestering agent for divalent cations. Inulin suitable as startingmaterial in derivatisation reactions should preferably have a({overscore (DP)}) of at least 20, whereas its level of low molecularweight sugars should be very low. Standard grade inulin from chicory orJerusalem artichoke has a too low ({overscore (DP)}) and a too highlevel of low molecular weight sugars, which makes derivatisation of saidinulins difficult.

To prepare inulin which is low in mono- and disaccharides and has a highaverage degree of polymerisation, preferably a ({overscore (DP)}) of atleast 20, various techniques have already been disclosed, for example, amethod of manufacture involving a directed crystallisation starting fromnative or standard grade chicory inulin as described in PCT patentapplication WO 96/01849. Inulin with such a profile is also verysuitable as ingredient in various food, feed, drinks and non-foodapplications, and as starting material for the manufacture ofhydrolysates and derivatives of inulin.

To prepare oligofructose, usually standard grade chicory inulin orpreferably chicory inulin with a higher ({overscore (DP)}), e.g. a({overscore (DP)}) of at least 20, is subjected to partial, enzymatichydrolysis, whereas to prepare fructose, typically in the form of afructose syrup, said inulins are subjected to complete enzymatic oracidic hydrolysis, as for example described in patent applicationsPCT/BE97/00087 and EP 97870111.8.

However, every treatment of native inulin or standard grade inulin toreduce the content of low molecular weight sugars, to increase the({overscore (DP)}) of the inulin, to modify the inulin profile,particularly to modify the distribution pattern of the inulin chains ofthe source inulin, or to transform inulin into oligofructose, requiresone or more additional processing steps, such as, for example, sizefractioning or hydrolysis. These additional process steps inevitablyresult in technical and economical disadvantages.

Accordingly, in the search for methods for producing inulin with apredetermined profile also an other approach is being prospected whichenvisages the direct production of inulin with a desired profile fromgenetically modified plants showing a modified inulin producing profile.

Herein the terms genetically modified, transformed and transgenic areused interchangeably; the terms 1-SST or 1-SST enzyme and 1-FFT or 1-FFTenzyme refer to the respective enzymes, whereas the terms sst103 orsst103 sequence and fft111 or fft111 sequence indicate an example of a1-SST, respectively 1-FFT encoding DNA sequence, and the terms sst103gene and fft111 gene indicate an example of a 1-SST, respectively a1-FFT enzyme encoding gene.

PCT patent application WO 96/01904 claims a method for producingfructo-oligosaccharides from a transgenic plant containing a geneconstruct comprising a fructosyl transferase encoding ftf gene fromStreptococcus mutans or a fructosyl transferase encoding Sac B gene fromBacillus subtilus or a mutated version of said genes. The invention aimsparticularly low molecular fructo-oligo-saccharides having a DP 3 to 4.

The patent application WO 96/01904 describes the isolation of an SSTenzyme from onion and shows the activity of the purified enzyme in vitroby incubation with sucrose with the formation of 1-kestose only. Neitherthe DNA sequence coding for this SST enzyme nor the amino acid sequenceof the purified SST enzyme were disclosed. The patent application alsodiscloses the sequences of two other fructosyl-transferases and the useof the 6-sft gene isolated from barley, where it is involved in thebiosynthesis of non-inulin type branched fructans, in an heterologousscreening leading to the isolation of the cDNA sequences of twovirtually identical genes from the flowers of onion, respectively pAC22and pAC92, which have been tested separately in protoplasts of tobacco.In the patent application (p. 19, line 27 to p. 20, line 2) it isindicated that in this way a fructosyltransferase activity could beshown but no experimental data were presented. Later experiments (Vijnet al., 1997) have revealed that pAC22 (designated pAC2 in Vijn et al.,1997) in fact encodes a 6-FFT enzyme (EMBL accession No Y07838)(fructan:fructan 6G-fructosyl transferase), which catalyses the transferof a fructosyl residue to the carbon 6 of the glucose moiety of sucrose,resulting in the formation of the trisaccharide neokestose (F2-6G1-2F)according to: GFF+GF→FGF+GF, so that, although presentingfructosyltransferase activity, the disclosed sequences of pAC22 andpAC92 thus do not represent 1-SST coding sequences.

On the one hand there is an essential difference between a 6-G-FFTenzyme and a 1-SST enzyme since (i) the 6-G-FFT enzyme can not usesaccharose as donor of a fructosyl residue (as a 1-SST enzyme does) butneeds kestose as a fructosyl unit donor, and (ii) the 6-G-FFT enzymecatalyses the synthesis of neokestose (FGF) constituting the startingmoiety for the building up of a fructan of the inulin neoseries class,whereas the 1-SST enzyme catalyses the synthesis of kestose (GFF)constituting the starting moiety for the building up of a fructan of theinulin class.

On the other hand there is an essential difference too between a 6-G-FFTenzyme and a 1-FFT enzyme, since the 6-G-FFT enzyme can catalyse onlythe production of polysaccharide chains with a low DP, i. e. a DP≦about10, whereas a 1-FFT enzyme can catalyse the synthesis of polysaccharidechains with a higher DP, i.e. a DP up to about 70 and even up to about100.

Said difference between the 6-G-FFT enzyme and the 1-SST enzyme,respectively the 1-FFT enzyme, is also reflected in the polysaccharidemolecules built up through catalysis by said enzymes. The polysaccharidechains built up via a 6G-FFT enzyme catalysis are of the inulinneoseries class, reach a DP of only up to about 10, have not a terminalglucose moiety, and are not strictly linear as a result of the 16disubstituted glucose moiety in the molecule, whereas the polysaccharidemolecules built up via 1-SST enzyme and 1-FFT enzyme catalysis can reacha DP of up to about 70, even up to about 100, have a terminal glucosemoiety, and present an essentially linear structure. Furthermore, theuse of the individual sequences in different DNA constructs to producetransgenic plants, in particular different crops, is mentioned but noexperimental data on the oligosaccharides obtained were given.Furthermore, Vijn et al., 1997 disclosed that introduction of the onion6-G-FFT enzyme encoding sequence in chicory resulted in a transgenicplant which made linear inulins (i.e. genuine chicory inulin) and inaddition fructans of the inulin neoseries. Apparently the native 1-SSTenzyme encoding sequences together with the native 1-FFT enzyme encodingsequences of the chicory ensured via the corresponding enzymes thesynthesis of the native inulin molecules, whereas the native 1-SSTenzyme encoding sequences together with the onion 6G-FFT enzyme encodingsequence lead via the corresponding enzymes to the synthesis of inulinneoseries of low molecular weight. However, a combination in the genomeof one and the same transgenic plant of a 1-SST encoding DNA sequenceand a 1-FFT encoding DNA sequence, wherein either or both of saidsequences are part of a recombinant construct, which combination isensuring via the respective enzymes the production of inulin with amodified inulin profile (the host plant being an inulin or a non-inulinproducing plant), has not been disclosed yet.

PCT patent application WO 96/21023 discloses a 1-SST encoding DNAsequence and a 1-FFT encoding DNA sequence both from J. artichoke,designated sst103 (or pSST103 when referring to the original clone beingthe sst103 inserted in the pBluescript SK vector) and fft111 (or pFFT111when referring to the original clone being the fft111 inserted in thepBluescript SK vector), respectively, the construction of recombinantgenes comprising said 1-SST enzyme or 1-FFT enzyme encoding sequence,and the transformation of plants (petunia and potato plants) byinsertion of said sst103 gene or fft111 gene in the plant genome.Expression of the sst 103 gene in the transgenic plants was shown byanalysis of the carbohydrate composition of the plants, revealing thepresence of fructo-oligosaccharides with a DP up to 5. Expression of thefft111 gene in the transgenic plants was demonstrated in vitro on thebasis of the ability of an extract of the transgenic plant to catalysethe synthesis of a polysaccharide G−(F)_(n) (n>4) at the expense ofsubmitted G−(F)₄ (G=glucosyl; F=fructosyl). No fructans were formed inthe latter plants because the FFT enzyme needs fructo-oligosaccharides(such as FOS with DP of 3 or 4) for the synthesis of oligofructans andfructans with a higher DP. Based on the above, the patent applicationclaims a method for producing a transgenic plant showing a modifiedinulin profile. A combination of DNA constructs comprising the 1-SSTencoding DNA sequence, respectively the 1-FFT encoding DNA sequence, inone and the same transgenic plant has not been disclosed.

In food, feed, drinks and non-food applications, as well as for themanufacture of fructo-oligosaccharides, fructose and various derivativesof inulin, industry is increasingly making use of inulin. As a resultthereof industry is continuously confronted with problems regarding thesupply of inulin, particularly the supply of inulin compositions withdesirable inulin profiles of highly linear polysaccharide chains. Saidinulin compositions should preferably be readily and directly producibleat industrial scale from plant sources at economically interestingcosts. Preferably said production should be possible by conventionalmanufacturing processes and without additional process steps to modifythe inulin profile of the native inulin, since each additional processstep would inevitably increase the manufacturing costs and reduce theoverall yield of suitable inulin.

OBJECT OF THE INVENTION

The object of the present invention is, inter alia, to provide a methodby which said and other problems can be solved, as well as to providemeans for use in said method.

SUMMARY OF THE INVENTION

As indicated above, in WO 96/21023 a 1-SST encoding DNA sequence, termedsst103 , from the genome of J. artichoke is disclosed which codes for a1-SST that catalyses in a plant the conversion of sucrose into kestose,thus enabling the plant to synthesise kestose (GFF), the smallest inulinmolecule, from sucrose.

The inventors have now discovered the existence of a further 1-SSTencoding DNA sequence in the genome of J. artichoke which apparently ispart of a sleeping gene of said genome. The sequence has been identifiedin the form of its cDNA by DNA sequencing and this cDNA sequence, termeda33, is given in SEQ ID NO: 1 and is shown in FIG. 1 as A33. In FIG. 1also the corresponding amino acid sequence is indicated, given in SEQ IDNO: 2.

Furthermore, the inventors have been able to identify a 1-FFT encodingDNA sequence in the genome of chicory and to identify its DNA sequencein the form of its cDNA by DNA sequencing. This cDNA sequence termedc86b, is given in SEQ ID NO: 3 and is shown in FIG. 2A as C86B, togetherwith the corresponding amino acid sequence, given in SEQ ID NO: 4.

The inventors have found that the a33 cDNA sequence can be expressed ina host organism, particularly a plant or a plant part, to produce active1-SST, when it is inserted in reading frame in a proper DNA construct inthe genome of the organism. Said construct typically comprises the a33cDNA sequence operably linked in the normal orientation to a promotersequence and to a terminator sequence which are active in said hostorganism. When the host organism is a plant, the construct is preferablyfurther comprising an operably linked DNA sequence encoding a targetingsignal or a transit peptide which ensures targeting of the a33 encoded1-SST enzyme to a specific subcellular compartment. The constructs, thusconstituting a 1-SST enzyme encoding recombinant gene (in short herein1-SST a33 gene), can be introduced into the genome of the host organismby conventional techniques.

Besides, the inventors have surprisingly found that said a33 cDNAsequence codes for an 1-SST enzyme which catalyses not only thetransformation of sucrose to kestose and to lower oligofructoses(DP≦about 5), as do the known 1-SST enzyme encoding DNA sequences suchas e.g. sst103 from J. artichoke, but encodes an 1-SST enzyme which isalso able to catalyse the synthesis of higher fructo-oligosaccharides(DP up to about 10) in a plant. The a33 cDNA thus encodes in fact a1-SST enzyme which has an activity which extends beyond the one of theknown 1-SST enzymes, e.g. the 1-SST enzyme encoded by the sst103 cDNA,and has also an activity which is with respect to the building up ofinulin chains functionally comparable to an aspect of a 1-FFT enzyme.

The inventors have also found that the c86b cDNA sequence can beexpressed in a host organism, particularly a plant or a plant part, toproduce active 1-FFT, when it is inserted in reading frame in a properDNA construct in the genome of the host organism. Said constructtypically comprises the c86b cDNA sequence operably linked in the normalorientation to a promoter sequence and to a terminator sequence whichare active in said host organism. When the host organism is a plant, theconstruct is preferably further comprising an operably linked DNAsequence encoding a targeting signal or a transit peptide which ensurestargeting of the c86b encoded 1-FFT enzyme to a specific subcellularcompartment. The constructs, thus constituting a 1-FFT enzyme encodingrecombinant gene (in short herein 1-FFT86b gene), can be introduced intothe genome of the host organism by conventional techniques. Expressionof said recombinant gene in a host plant results in the production of1-FFT, which catalyses, as mentioned above, the stepwise synthesis ofinulin by transformation of an oligofructose chain or inulin chain intoan inulin molecule with a higher DP.

Furthermore, the inventors have found that a host organism, inparticular a plant, can be transformed to comprise in its genome acombination of one or more expressible 1-SST enzyme encoding genes andone or more expressible 1-FFT enzyme encoding genes, wherein either the1-SST enzyme encoding genes or the 1-FFT enzyme encoding genes or bothcomprise one or more recombinant genes containing one or more 1-SSTenzyme encoding DNA sequences, respectively one or more 1-FFT enzymeencoding DNA sequences, of plant origin, resulting in a transgenicorganism, particularly a transgenic plant, with a modified inulinproducing profile.

Moreover, the inventors have found that the inulin producing profile ofa plant and the profile of the inulin produced by a plant can bemodified and even controlled, i.e. modified to yield a desired inulinprofile, by producing a transgenic plant which comprises in its genome acombination of one or more expressible 1-SST enzyme encoding genes andone or more expressible 1-FFT enzyme encoding genes which code for 1-SSTand 1-FFT enzymes with different kinetic properties. Either the 1-SSTenzyme encoding genes or the 1-FFT enzyme encoding genes or bothcomprise one or more recombinant genes containing one or more 1-SSTenzyme encoding DNA sequences, respectively one or more 1-FFT enzymeencoding DNA sequences, from plant sources, the latter 1-SST encodingsequences and 1-FFT encoding sequences being from the same or fromdifferent plant sources.

On the basis of said findings, the inventors have been able to provide asolution to the above mentioned and other problems by the presentinvention.

Accordingly, in a first embodiment, the invention relates to a methodfor producing a transgenic plant by transforming a host plant tocomprise in its genome a combination of one or more expressible 1-SSTenzyme encoding genes and one or more expressible 1-FFTenzyme encodinggenes, wherein either the 1-SST enzyme encoding genes or the 1-FFTenzyme encoding genes or both comprise one or more expressiblerecombinant genes containing one or more expressible 1-SST enzymeencoding DNA sequences, respectively one or more expressible 1-FFTenzyme encoding DNA sequences which are of plant origin, comprisingtransforming a host plant by inserting one or more of said 1-SST enzymeencoding genes and/or one or more of said 1-FFT enzyme encoding genesinto the genome of the host plant, yielding a transgenic plant with amodified inulin producing profile.

In accordance with the present invention, said 1-SST encoding DNAsequence(s) and said 1-FFT encoding DNA sequence(s) of said recombinantgenes are not restricted to the sequences as obtained from the plantsources, but also include expressible homologous sequences thereof witha degree of homology of at least 70%, preferably at least 75%, morepreferably at least 80% even more preferably at least 85%, and mostpreferably at least 90%, respectively, irrespective whether or not thehomologous sequences are derived from plant sources or are obtained bymutagenesis of DNA sequences from plant sources or from micro-organisms.

By inulin is meant herein fructans of the inulin class, i.e. moleculesof the general formulae GF_(n) and F_(m), as defined above, exclusive offructans of the inulin neoseries class corresponding to the generalformula

 F2−(1F2)m′−6G1−(2F1)n′−2F

wherein F and G respectively represent fructose and glucose, and m′ andn′ represent integers which can be the same or different.

By modified inulin producing profile is meant the production of inulinby a transgenic plant which is quantitatively and/or qualitativelydifferent from the production of inulin by the non-transformed hostplant.

By modified inulin profile is meant an inulin profile which isqualitatively different from the one of the inulin produced by the hostplant, i.e. an inulin composition wherein the ratio of monosaccharides,disaccharides, oligo-saccharides and/or the distribution pattern of thechain length of the individual inulin molecules, i.e. the DP and the({overscore (DP)}), are different from the ones of the inulincomposition produced by the non-transformed host plant. For the sake ofconvenience, the term modified inulin producing profile is embracingherein a modified inulin producing profile, a controlled inulinproducing profile as well as a modified inulin profile and a controlledinulin profile.

The non-transformed host plant suitable for the invention can be aninulin producing plant containing in its genome one or more 1-SSTencoding genes and 1-FFT encoding genes or only 1-SST encoding genes, ora non-inulin producing plant.

If in the genome of the host plant a 1-FFT encoding gene and/or a 1-SSTencoding gene is present, when producing the desired transgenic plantaccording to the present invention said gene or genes can be maintainedor their expression can be totally or partially suppressed by knowntechniques. For example the expression of said genes can be suppressedthrough anti-sense expression or co-suppression strategies.

The said 1-SST enzyme encoding genes, respectively the said 1-FFT enzymeencoding genes, of the genome of the transgenic plant in accordance withthe present invention, can consist of a native gene or a mixture ofdifferent native genes, of a mixture of native and recombinant genes, orof one or more different recombinant genes.

If said combination of 1-SST encoding gene(s) and 1-FFT encoding gene(s)comprises known 1-SST encoding gene(s), respectively known 1-FFTencoding gene(s), these known genes may be the ones which are present inthe genome of the non-transformed host plant.

If said combination comprises recombinant genes, their 1-SST enzymeencoding sequence, respectively the 1-FFT enzyme encoding sequence, canbe a known one or a novel one, from a plant source, or a homologoussequence thereof, as defined above, which encodes a 1-SST enzyme,respectively a 1-FFT enzyme.

If both the 1-SST enzyme encoding genes and the 1-FFT enzyme encodinggenes comprise a recombinant gene, the 1-SST enzyme encoding sequence(s)and the 1-FFT enzyme encoding sequence(s) of said recombinant genes canbe from the same or from different plant species.

If more than one 1-SST enzyme encoding DNA sequence, respectively morethan one 1-FFT enzyme encoding DNA sequence, is present in the genome ofthe transgenic plant, the 1-SST encoding sequences, respectively the1-FFT encoding sequences, may be identical or not, may be present in oneor in different genes, and these 1-SST encoding genes, respectively1-FFT encoding genes, may be present on one or on different chromosomes.

In a preferred embodiment of the invention, the recombinant 1-SSTencoding gene comprises said a33 cDNA sequence or an expressiblehomologous sequence thereof as defined above.

In another preferred embodiment, the recombinant 1-FFT encoding genecomprises said c86b cDNA sequence or an expressible homologous sequencethereof as defined above.

In a further preferred embodiment, the recombinant 1-SST encoding genecomprises said a33 cDNA sequence or said homologous sequence and the1-FFT encoding gene comprises said c86b cDNA sequence or said homologoussequence.

Said homologous sequences are at least 70% identical to said a33 cDNA,respectively to said c86b cDNA, irrespective of whether or not thehomologous sequences are derived from another plant species or areobtained by mutagenesis of fructosyltransferase-encoding sequences fromplant sources or from micro-organisms. Preferably the degree of homologyis at least 75%, more preferably at least 80%, and even more preferablyat least 85%. Most preferably the degree of homology is at least 90%.

In a further preferred embodiment, the transgenic plant has beenproduced by inserting into the genome of a host plant a combination ofone or more genes with a known 1-FFT enzyme encoding DNA sequence andone or more genes with the 1-SST enzyme encoding a33 cDNA sequence orsaid respective homologous sequences thereof which encode a 1-FFTenzyme, respectively, a 1-SST enzyme. In another preferred embodiment,the transgenic plant has been produced by inserting into the genome of ahost plant a combination of one or more genes with a known 1-SSTencoding DNA sequence and one or more genes with the 1-FFT enzymeencoding c86b cDNA, or said respective homologous sequences thereof. Ina further preferred embodiment, the transgenic plant has been producedby inserting into the genome of a host plant a combination of one ormore genes with the 1-SST enzyme encoding a33 cDNA sequence or a saidhomologous sequence thereof and one or more genes with the 1-FFT enzymeencoding c86b cDNA or a said homologous sequence thereof. In a stillfurther preferred embodiment of said execution forms of the invention,the host plant is a non-inulin producing plant.

Typical combinations of 1-SST encoding genes and 1-FFT encoding genesaccording to the invention comprise respectively a 1-SST enzyme encodingsequence or a said homologous sequence thereof and a 1-FFT enzymeencoding sequence or a said homologous sequence thereof selected fromplant species of the Asteraceae family, with the 1-SST encoding sequenceand the 1-FFT encoding sequence being selected from the same or fromdifferent plant species.

Further typical combinations of 1-SST encoding genes and 1-FFT encodinggenes according to the invention comprise respectively a 1-SST enzymeencoding sequence or a said homologous sequence thereof and a 1-FFTenzyme encoding sequence or a said homologous sequence thereof selectedfrom plant species from the same or different plant families of thegroup consisting of the Asteraceae (Compositae) and Campanulaceae,comprising plant species such as, for example, Echinops, species,Helianthus tuberosus, Cichorium intybus, Dahlia species, Cynara species,Viguiera species, such as e.g. Viguiera discolor, Viguiera deltoida,Viguiera annua, Viguiera lanata, Viguiera multiflora, Veronia herbacea,Scorzonera hispanica, Tragopogon porriflorus, Taraxacum, species,Arctium lappa, Campanula rapuncoloides and Bellis perennis.

Typically suitable DNA sequences include, for example, the 1-SSTencoding sequences sst103 (J. artichoke), a33 (J. artichoke), c33(chicory), Genbank accession No U81520 (chicory), Genbank accession NoY09662 (Cynara scolymus), and the 1-FFT encoding sequences c86b(chicory) and fft111 (J. artichoke).

By the selection of a proper combination of one or more of saidexpressible 1-SST encoding genes and one or more of said expressible1-FFT encoding genes, in combination with the selection of a properratio between the expression of the 1-SST encoding and 1-FFT encodinggenes and the selection of a suitable host plant, a transgenic plant canbe produced according to the invention with a desired modified inulinproducing profile. In fact, the selection of proper combinations of saidparameters by routine experiments, makes it possible to produce inulinwith an almost tailored profile and to modify in a desired manner theinulin producing profile of a given host plant.

In a preferred embodiment, the genome of the transgenic plant comprisesa combination of said expressible 1-SST encoding genes and saidexpressible 1-FFT encoding genes which induces the synthesis of nativeinulin with a degree of polymerisation which is higher, respectivelylower, than the one of the native inulin produced, if any, by thenon-transformed host plant.

When an inulin essentially composed of fructo-oligosaccharides withinulin chains with a DP≦about 10 is desired, a method is providedaccording to a particular embodiment of the present invention, forproducing a transgenic plant which produces inulin with such profile.This is very advantageous because no conventional, partial hydrolysisstep of inulin with longer carbohydrate chains, such as e.g. standardgrade chicory inulin with a ({overscore (DP)}) of about 10, needs to beincluded in the production process of said inulo-oligosaccharides.Accordingly, in a first variant, a method is provided for producing atransgenic plant by transforming a host plant to comprise in its genomea combination of one or more expressible 1-SST encoding genes,originating from the host plant or from a different plant source and oneor more 1-SSTa33 genes. If the host plant contains a native 1-SSTencoding gene and a native 1-FFT encoding gene in its genome, theexpression of the native 1-FFT encoding gene or of both the native 1-SSTencoding gene and the native 1-FFT encoding gene can optionally besuppressed by known techniques, for example through anti-senseexpression. In a second variant, a transgenic plant is produced byinserting into the genome of a host plant which does not contain a 1-SSTencoding gene nor a 1-FFT encoding gene, one or more 1-SST a33 genes orgenes which contain a cDNA sequence which is an homologous sequencethereof, as defined above, which encodes an a33 1-SST enzyme. Thisparticular embodiment of the present invention has become possible as aresult of the fact that the 1-SST a33 gene codes for an enzyme whichpresents an activity of a 1-SST enzyme, i.e. catalysing the synthesis ofkestose from sucrose, but also presents a moderate activity with respectto the building up of inulin chains which is comparable to an aspect ofa 1-FFT enzyme activity, i.e. catalysing the synthesis offructo-oligosaccharides from kestose or fructo-oligosaccharides with alow DP.

The selection of the optimal combination and ratio of the number ofconcerned 1-SST and 1-FFT enzyme encoding DNA sequences in combinationwith the selection of the most suitable plant species for the productionof a desired inulin profile can be made by the skilled person accordingto conventional techniques, for example through routine experiments.

In the method according to the invention, the transgenic plant can beproduced by inserting into the genome of the host plant by conventionaltechniques one or more expressible 1-SSTa33 genes or expressiblehomologues thereof, or one or more of said expressible 1-SST encodinggenes from plant sources or said homologues thereof and/or one or moreof said expressible 1-FFT encoding genes from plant sources or saidexpressible homologues thereof, resulting in a transgenic plant whichcomprises in its genome one or more 1-SSTa33 enzyme encoding genes or acombination of said 1-SST encoding genes and said 1-FFT encoding genesas defined herein above.

In a typical execution form, a cell of a host plant is transformed tocomprise in its genome a said combination of 1-SST encoding genes and1-FFT encoding genes as defined above, by inserting by conventionaltechniques one or more of said genes into the genome of the cell,followed by regenerating a transgenic plant from said transformed cell.If the host plant is transformed with both said 1-SST encoding genes andsaid 1-FFT encoding genes, the genes can be inserted into the hostgenome simultaneously or in subsequent steps.

In a typical execution form, said method comprises the followingsubsequent steps which can be carried out by conventional techniques:

i) the preparation of a recombinant gene construct comprising one ormore 1-SST enzyme encoding DNA sequences, respectively 1-FFT enzymeencoding DNA sequences as defined above, operably linked to a promotersequence active in said host plant and a terminator sequence active insaid host plant,

ii) introduction of the recombinant gene construct obtained in step i)into the genome of a cell of the host plant, and

iii) regeneration of the transformed plant cell obtained in step ii) tothe corresponding transgenic plant.

More specifically the method of the invention comprises preferably thefollowing subsequent steps:

a) the construction of a recombinant gene, i.e. a recombinant DNAconstruct, comprising essentially the following sequences:

1. a promoter which ensures the formation of a functional RNA or afunctional protein in the intended target plant, target organs, tissuesor cells thereof,

2. one or more copies of a DNA sequence encoding respectively 1-SST or1-FFT enzyme, functionally connected to said promoter,

3. a transcription terminator operationally connected to said 1-SST or1-FFT enzyme encoding DNA sequence,

4. a DNA sequence encoding a targeting signal or a transit peptide whichensures targeting of the 1-SST enzyme, respectively the 1-FFT enzyme toa specific subcellular compartment,

b) the introduction of the recombinant gene obtained in a) into thegenome of the host plant yielding genetically modified material,typically a cell, comprising said combination of 1-SST enzyme and 1-FFTenzyme encoding sequences, and regeneration of the geneticallytransformed material in the corresponding transformed host plant.

In the recombinant DNA construct according to the present invention, oneor more copies of the 1-SST and 1-FFT enzyme encoding DNA sequences arepreferably linked to one or more regulatory sequences which areoperational in the host plant ensuring proper expression of said DNAsequence at a sufficiently high expression level in the host plant, inthe different plant organs, tissues or cells. Regulatory sequences are,for example, a promoter, a termination signal and a transcription ortranslational enhancer. A promoter can, for example, be the 35S promoterof the cauliflower mosaic virus (CaMV) or an organ-specific promoterlike the tuber-specific potato proteinase inhibitor II promoter, or anyother inducible or tissue-specific promoter.

The production of inulin being particularly advantageous in organsstoring large amounts of sucrose, such as the tap roots of sugar beet orthe stems of sugar cane, a highly preferred promoter is a promoter whichis active in organs and cell types which normally accumulate sucrose(the primary substrate for inulin synthesis).

The production of inulin is particularly advantageous in the vacuolewhich can accumulate very high concentrations of sucrose (e.g. up toabout 500 mol m⁻³). Accordingly, in the recombinant DNA according to thepresent invention, the DNA sequence encoding 1-SST or 1-FFT enzyme ispreferably linked to a sequence encoding a transit peptide which directsthe mature 1-SST or 1-FFT enzyme protein to a subcellular compartmentcontaining sucrose, such as for example said vacuole.

In a preferred embodiment, the host plant is a non-inulin producingplant; in another preferred embodiment, the host plant is an inulinproducing plant.

Typical host plants for use in the method according to the invention areplants which can easily be grown, which are rather resistant to attackby injurious organisms such as insects and fungi, which give a highyield of plant material per hectare and which can be easily harvestedand processed. Besides, said plants should after transformation be ableto produce large amounts of inulin, give a high content of producedinulin based on fresh plant material and preferably be able to depositsaid inulin in a concentrated manner in parts of the plant, preferablyin tap roots or tubers, which can be easily harvested, stored andprocessed.

Other typical host plants for use in the method of the invention arenon-inulin producing plants which are quite sensitive to abioticstresses such as, for example, drought, cold, and other ones.Transformation of said host plants according to the present inventionresulting in a transgenic plant which is producing inulin, even in aquite low level, may significantly increase the resistance of the plantagainst said abiotic stresses, particularly against drought and/or cold.

Typical host plants suitable for use in the method according to theinvention include corn, wheat, rice, barley, sorghum, millets,sunflower, cassava, canola, soybean, oil palm, groundnut, cotton, sugarcane, chicory, bean, pea, cow pea, banana, tomato, beet, sugar beet,Jerusalem artichoke, tobacco, potato, sweet potato, coffee, Gocoa andtea.

In a further embodiment, the present invention provides a method formodifying the inulin producing profile of a plant and for controllingthe profile of the inulin produced by a plant, which comprisesgenetically transforming a plant by inserting into its genome one ormore expressible 1-SSTa33 genes or expressible homologues thereof, orone or more expressible 1-SST encoding genes, and/or one or moreexpressible 1-FFT encoding genes, as defined above, yielding atransgenic plant comprising in its genome a combination of said 1-SSTencoding genes and said 1-FFT encoding genes, as defined herein above,which plant, when cultured under conventional conditions, shows amodified inulin producing profile.

In still another embodiment, the present invention relates to a methodfor producing inulin from plant material, particularly inulin with amodified profile, by conventional techniques, wherein the source plantmaterial for the method is material, typically tap roots or tubers, froma transgenic plant which comprises in its genome one or more expressible1-SST a33 enzyme encoding genes, or a combination of one or moreexpressible 1-SST encoding genes and one or more expressible 1-FFTencoding genes, or expressible homologues of said genes, as definedabove. Such transgenic plant is obtainable by a method of the presentinvention as described herein before.

The production of inulin from plant parts is well known in the art andcommonly comprises (i) an isolation step, wherein the crude, nativeinulin is isolated from the plant material (typically involvingextraction of shredded plant parts e.g. tap roots or tubers, with warmwater ), followed by (ii) a purification step, typically comprising adepuration treatment (involving liming and carbonatation or anotherflocculation technique and filtration) followed by a refining treatment(involving treatment over ion-exchangers, treatment with active carbonand filtration) and optionally a concentration of the purified inulinsolution, and (iii) an isolation step wherein the inulin is isolated inparticulate form from the purified inulin solution obtained in (ii), forexample by spray-drying or by directed crystallisation, filtration anddrying.

The invention further embraces the novel DNA sequences a33 cDNA and c86bcDNA, and homologous sequences thereof, as defined above, encodingrespectively 1-SST enzyme and 1-FFT enzyme, as well as novel recombinantDNA constructs, novel recombinant genes and vectors comprising one ormore of said novel cDNA sequences. Said novel cDNA sequences aresuitable intermediates for the construction of said novel recombinantDNA constructs and recombinant genes, which are in turn, suitableintermediates and tools for the production of transgenic plantspresenting a modified inulin producing profile according to the presentinvention.

The invention furthermore embraces a novel combination of one or moreexpressible 1-SST encoding genes and one or more expressible 1-FFTencoding genes, wherein either the 1-SST encoding genes or the 1-FFTencoding genes or both comprise one or more recombinant genes of whichone or more of the 1-SST enzyme encoding DNA sequences and one or moreof the 1-FFT enzyme encoding DNA sequences are of plant origin orhomologues thereof, as defined above. Said novel combination of genesconstitutes a suitable intermediate and tool for the production oftransgenic plants according to the present invention.

The invention also embraces a transgenic plant, producible by a methodaccording to the present invention, the genome of which comprises acombination of one or more expressible 1-SST encoding genes and one ormore expressible 1-FFT encoding genes, as defined above.

The present invention also includes cells, plant tissue, plant parts,roots and shoots of transgenic plants according to the invention as wellas seeds thereof, which comprise in their genome a combination of one ormore expressible 1-SST encoding genes and one or more expressible 1-FFTencoding genes, as defined above.

The present invention also includes novel inulin compositions, i.e.inulin having a novel inulin profile, obtainable by a method accordingto the present invention, and the use thereof in the manufacture offood, feed, drinks, and non-food compositions, of derivatives of inulin, and of partial and complete hydrolysates of inulin.

In still a further embodiment, the present invention relates to apurified and isolated polypeptide having an amino acid sequence as shownin SEQ ID NO:2, respectively in SEQ ID NO: 4, and to purified andisolated respective homologues thereof having at least 70% homology,preferably at least 75%, more preferably at least 80%, even morepreferably at least 85%, and most preferably at least 90% homology, aswell as to a fragment of said polypeptides, which polypeptides, saidhomologues thereof and said fragments thereof have 1-SST, respectively1-FFT, activity.

Said polypeptides, homologues and fragments can be obtained according toconventional techniques. For example the polypeptides of SEQ ID NO: 2and of SEQ ID NO: 4 can be obtained from chicory roots through apurification procedure based on ammonium sulphate precipitation,followed by lectin affinity chromatography, anion- and cation exchangechromatography.

In an ultimate embodiment, the present invention relates to antibodiescapable of specifically binding one or more polypeptides and/orhomologues and/or fragments thereof as defined above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: shows the nucleotide sequence (SEQ ID NO: 1) (A33) and deducedamino acid sequence (SEQ ID NO: 2) of the isolated a33 cDNA

FIG. 2A: shows the nucleotide sequence (SEQ ID No. 3) (C86B) and deducedamino acid sequence (SEQ ID No. 4) of the isolated c86b cDNA.

FIG. 2B: shows the nucleotide sequence (SEQ ID No. 5) (C33) and deducedamino acid sequence (SEQ ID No. 6) of the isolated c33 cDNA.

FIG. 3: depicts a Southern blot analysis of Helianthus tuberosus genomicDNA probed with an a33 PCR fragment. H. tuberosus DNA was digested withEcoR1, EcoR5, XbaI, AflIII, DraI, NdeI, HincII and, XhoI. Plasmid pA33(the a33 cDNA in the pBluescript vector) was used as a positive control,potato DNA digested with EcoRI was used as negative control.

FIG. 4: depicts a Northern blot analysis of RNA isolated from varioustissues from Helianthus tuberosus.: RNA was isolated from dormant tubers(lane 1), sprouting tubers (lane 2), stolons (lane 3), tubers with a 2-5mm diameter (lane 4), tubers with a 2-2.5 cm diameter (lane 5), tuberswith a 5-7 cm diameter (lane 6), leaves (lane 7), stems (lane 8),fibrous roots (lane 9), receptacle (lane 10) and flower (lane 11). RNAwas probed with an sst103 (FIG. 4A), fft111 (FIG. 4B) or a33 PCRfragment (FIG. 4C).

FIG. 5: presents the chimeric gene construct pA33.236 consisting of theenhanced CaMV35S promoter (Penh35S) with a ALMV translational enhancer(amv) , the coding sequence of a33 (a33) and the nos-termination signal(Tnos).

FIG. 6.: shows a TLC analysis of leaves and tubers of transgenic potatoharbouring the pA33.236 construct. TLC plates were developed twice in90% aqueous acetone., G=glucose standard, F=fructose standard GF=sucrosestandard, GF₂ is the GF₂ standard, H.t.=standard fructan mixture frommature tubers of H. tuberosus. Plant no 27B, 50, 83, 93 and 23 representindividual potato plants harbouring the pA33.236 construct. Control is acontrol plant harbouring the AGL0 construct. Form each plant (controlplant as well as transgenic plants) two tubers were analysed (lanes 1and 2) and one leaf (lane 3).

FIG. 7: shows HPAEC separations of carbohydrates extracted from leavesof control potato transformed with AGL0 lacking the binary vector (A),from leaves of transgenic potato harbouring the pA33.236 construct (B)and a standard inulin extracted from chicory tap roots (C),

FIG. 8: represents the chimeric gene construct pUCPA33.342 consisting ofthe coding sequence of a33 (a33) under control of the enhanced CaMV35Spromoter (Penh35S) and the nos-termination signal (Tnos), the ALMVtranslational enhancer (amv), and the herbicide resistance gene (pat)under control of the promoter (P35S) and terminator sequences (T35S) ofthe CaMV35S gene.

FIG. 9: represents the chimeric gene construct pUCPCA342.25 harbouringthe coding sequences of a33 (a33) and c86b (c86b), each under control ofthe enhanced CaMV35S promoter (Penh35S) and the nos terminator (Tnos),the ALMV translational enhancer (amv), and the herbicide resistance gene(pat) under control of the promoter (P35S) and terminator sequences(T35S) of the CaMV35S gene.

FIG. 10: represents the chimeric gene construct pUCPSF344.18 harbouringthe coding sequences of fft111 (1-fft) and sst103 (1-sst), each undercontrol of the enhanced CaMV35S promoter (Penh35S) and thenos-termination signal (Tnos), the ALMV translational enhancer (amv),and the herbicide resistance gene (pat) under control of the promoter(P35S) and terminator sequences (T35S) of the CaMV35S gene.

FIG. 11: represents the chimeric gene construct pUCPSC344.14 harbouringthe coding sequences of c86b (c86b) and sst103 (1-sst), each undercontrol of the enhanced CaMV35S promoter (Penh35S) and thenos-termination signal (Tnos), the ALMV translational enhancer (amv),and the herbicide resistance gene (pat) under control of the promoter(P35S) and terminator sequences (T35S) of the CaMV35S gene.

FIG. 12: represents the chimeric gene construct pUCPAF21.17 harbouringthe coding sequences of a33 (a33) and fft111 (1-fft), each under controlof the enhanced CaMV35S promoter (Penh35S) and the nos-terminationsignal (Tnos), the ALMV translational enhancer (amv), and the herbicideresistance gene (pat) under control of the promoter (P35S) andterminator sequences (T35S) of the CaMV35S gene.

FIG. 13: depicts a Northern blot analysis of RNA isolated from leaves ofsugar beets: RNA was isolated from transgenic sugar beet line 7PSF22(lane 1), transgenic line 9PSC2 (lane 2), transgenic line 8PAF1 (lane3), transgenics line 8PAF5 (lane 4) and a non-transgenic control sugarbeet (lane 5). The RNA was probed with DNA fragments specific forsst103, a33, c86b or fft111.

FIG. 14: shows HPAEC separations of carbohydrates extracted from taproot of transgenic sugar beet line 7PSF22 expressing the sst103 andfft111 genes.

FIG. 15: shows HPAEC separations of carbohydrates extracted from taproot of chicory.

FIG. 16: shows HPAEC separations of carbohydrates extracted from taproot of transgenic sugar beet line 9PSC2 expressing the sst103 and c86bgenes.

FIG. 17: shows HPAEC separations of carbohydrates extracted from taproot of transgenic sugar beet line 8PAF5 expressing the a33 and fft111genes.

FIG. 18: shows HPAEC separations of carbohydrates extracted from taproot of transgenic sugar beet line 8PAF1 expressing the a33 and fft111genes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention results in significant technical and economicaladvantages. At first, the invention provides novel plant sources ofinulin thus helping to solve the problem of sufficiency of inulinsupply. Said plant sources may comprise known inulin producing plantswhich as a result of a genetically modification according to theinvention produce more inulin and/or inulin of a preferred modifiedprofile entailing improved functional properties, as well as non-inulinproducing plants which have been transformed to produce inulin,particularly inulin having a desired profile. Secondly, the methodaccording to the present invention enables to obtain directly, i.e.without any additional process steps such as e.g. size fractioning orpartial hydrolysis, inulin with a certain desirable inulin profile, suchas inulin essentially composed of inulin chains with a DP≦10. Thisobviously has considerable advantages with respect to manufacturing andmanufacturing costs.

Furthermore, the invention provides the possibility of producing manytransgenic plant species and variants thereof within the scope of thepresent invention, resulting from the flexibility of the method of thepresent invention for producing a transgenic plant with a modifiedinulin producing profile. Indeed, the possibility to produce manydifferent transgenic plants, starting from different host plant speciesand by using various different combinations of the 1-SST and/or 1-FFTenzyme encoding sequences according to the invention, i.e. includingdifferent 1-SST, respectively 1-FFT enzyme encoding DNA sequencesoriginating from various different plant species or homologous sequencesthereof, as defined above, and different ratio's of said 1-SST,respectively 1-FFT enzyme encoding DNA sequences, enables to producevarious inulin compositions with a different, desired profile. Saiddesired inulin profile is often translated in improved functionalproperties of the inulin and commonly in improved physico-chemicaland/or organoleptic properties of end products containing said inulin,and of hydrolysates and of derivatives of said inulin.

The invention enables furthermore to produce transgenic plant specieslike for example cultural crops and ornamental plants with an increasedresistance against abiotic stresses like e.g. drought and/or cold, whichalso results in economically important advantages.

The polypeptides, homologues and fragments thereof, as defined above,may be used to catalyse specific in vitro chemical reactions, such as,for example, the use of polypeptide of SEQ ID NO: 2 in the synthesis of1-kestose from sucrose by a fructosyl transfer reaction; the use ofpolypeptide of SEQ ID NO:4 in the synthesis of fructo-oligosaccharidesor inulin by a fructosyl transfer reaction from fructo-oligosaccharides;and the use of a combination of the polypeptides of SEQ ID NO:2 and SEQID NO:4, or respective homologues or fragments thereof as defined above,for the synthesis of fructo-oligosaccharides and inulin from one or morecarbohydrates selected from the group consisting of sucrose, 1-kestoseand oligofructoses.

The invention is further described in detail in the experimental partbelow, comprising the methodology and examples. It is emphasised thatthe examples are given as merely illustrative, non-limitative examplesof the invention.

DNA METHODOLOGY

DNA and RNA isolation, subcloning, restriction analysis and sequencingwere performed using standard methods described in molecular biologymanuals (e.g. Sambrook et al. 1989; Ausubel et al. 1994).

DNA and protein alignments were performed using the DNA analysissoftware Genworks 2.5.1. (Intelligenetics, Inc., CA). This program usesa progressive alignment method, building the alignment in approximatephylogenetic order using an algorithm similar to FASTA. Proteinalignments use a PAM-250 scoring matrix.

Construction and Screening of a cDNA Library from H. tuberosus

Ten μg of poly(A)⁺RNA isolated from tubers of Helianthus tuberosus‘Colombia’, harvested in July, was used as starting material for theconstruction of an Uni-ZAP XR cDNA library (Stratagene, La Jolla,Calif., USA). Construction, plating and screening of the library wereperformed according to the protocols developed by Stratagene (USA, cat.no. 237211). About 100.000 plaques of the unamplified cDNA library of H.tuberosus were blotted onto Hybond N⁺ (Amersham) membranes and screenedwith a probe comprising two DNA fragments. A 840 bp DNA fragment wassynthesised by PCR using primers 5′-TACGCTGTCAACTCGTCGG-3′ and5′-TTGAATAGAACATCGGGCTCTAGCG-3′ and plasmid pSST103 (sst103 cDNA inpBluescript phagemid, Van der Meer et al. 1998) as a template. A 860 bpDNA fragment was obtained by PCR using primers5′-GTTCAACGCTGCTTGATCCACC-3′ and 5′-ACCACGGTCCTTCCAAACGG-3′ and plasmidpFFT111 (fft111 cDNA in pBluescript phagemid Van de Meer et al., 1998)as a template. The two PCR fragments were radiolabelled by the RadPrimeDNA labelling system (Life Technologies). Hybridisation was at 50° C. in500 mM NaP-buffer, pH 7.2,7% SDS, 1% BSA, 1 mM EDTA. Filters were washedto a final stringency of 2×SSC, 0.1% SDS at 50° C. (2×30 min) andfinally exposed to X-omat AR (Kodak). Positive plaques were purified in34 rounds of plaque hybridisation. The pBluescript phagemids wereexcised from the Uni-ZAP vector using the Exassist/Solr system(Stratagene). The inserts were analysed by restriction enzyme analysisand sequencing.

Construction and Screening of a cDNA Library from Cichorium intybus

Ten μg of poly(A)⁺RNA isolated from tap roots of Cichorium intybus‘Cassel’, which was harvested in July, was used as starting material forthe construction of a lambda TriplEx cDNA library (Clontechlaboratories). Construction, plating and screening of the library wereperformed according to the protocols developed by Clontech (Palo Alto,Calif., cat. no. CS1010t). About 60.000 plaques of the unamplifiedchicory library were screened with a mixture of the two ³²P-labelled DNAprobes obtained by PCR and RadPrime labelling as described above.Hybridisation and washing of Hybond-N⁺ membranes were performed underlow stringency conditions (hybridisation at 50° C., final wash step with2×SSC, 0.1% SDS, 50° C.). Positive plaques were purified in 3-4 roundsof plaque hybridisation. The lambda Trip1Ex clones were converted intopTriplEx clones using the cre-recombinase mediated site-specificrecombination at the loxP sites flanking the embedded pTriplEx(Clontech). The pTriplEx clones were analysed by restriction enzymeanalysis and DNA sequencing.

PCR

PCR was performed in 50 μl PCR buffer (Life Technologies), containing100 pmol plasmid as a template, and 100 pmol of gene specific primers(specific for sst103, fft111, a33, c33, or c86b, depending on theexperiment). Amplification involved 30 cycles of denaturing (0.5 min,92° C.), annealing (1 min, 55° C.) and amplification (1 min, 72° C.).The resulting fragments were checked by DNA sequencing and restrictiondigestion to confirm the identity.

Transformation of Potato

Transformation of potato was performed according to Visser (1991), withthe following modifications. Stem internodes, cut from in vitro grownSolanum tuberosum, were placed in R₃B agar plates, on top of filterpaper, which was soaked with 2 ml PACM medium. The internodes wereincubated 24 h at 21° C. The binary plasmid pA33.236 was introduced intothe Agrobacterium tumefaciens strain ‘AGL0’ (Lazo et al. 1991) by adding0.5 microgram of plasmid DNA to 200 microliter of competentAgrobacterium cells. Competent cells were prepared according to Sambrook(1989). The plasmid DNA-Agrobacterium mixture was incubated on ice, for30 min, then frozen in liquid nitrogen and thawed in a water bath at 37°C. for 5 min. After addition of 1 ml YEP medium, the bacteria wereincubated at 28° C. for 2 hours with gentle shaking. Cells were pelletedand resuspended in 100 μl YEP-medium. Finally, transformed bacteria wereselected on YEP-agar plates containing 25 mg/l kanamycin. The presenceof pA33.236 was tested by restriction enzyme analysis.

A. tumefaciens cells, transformed with pA33.236 were grown at 30° C. in5 ml LB medium, containing 50 μg/ml kanamycin and 100 μg/ml rifampicin.After 48 h of growth, the cells were washed twice in 5 ml 2 mM MgSO₄,then suspended in 5 ml MS medium. Stem intemodes were incubated in 20 mlof a diluted (100× in MS) A. tumefaciens suspension, and gently shakenfor 30 min. After incubation, the stem internodes were blotted dry onfilter paper and placed on top of PCM-agar plates (48 h at 21° C.).Callus formation was induced by transferring the internodes to PCM agarplates, containing 200 mg/l cefotaxime and 100 mg/l kanamycine (96 h at21° C.). Shoot formation was induced at 21° C. by transferring the steminternodes to PSM-agar, containing 200 mg/l cefotaxime and 100 mg/lkanamycin. Regenerating explants were transferred to fresh medium everythree weeks. Root formation was induced by transferring the transgenicshoots to MS 30 medium, containing 200 mg/l cefotaxime and 50 mg/lkanamycine. Rooted plantlets of 5-10 cm were transferred to thegreenhouse.

DNA and RNA Analysis of Jerusalem Artichoke

Total DNA was isolated from mature young mature leaves which wereharvested 3 months after transfer of the plants to the greenhouse.Aliquots of the DNA was digested with a number of restriction enzymes.Total RNA was isolated from stolons, tubers of various ages, stems,leaves and flower tissues. After electrophoresis on 1% agarose, DNA orRNA was blotted onto Hybond-N⁺ (Amersham) and UV cross-linked. Filterswere hybridised with either an fft111, sst103 (see Construction andscreening of a cDNA library from H. tuberosus) or an a33 probe. A 1140bp DNA fragment specific for a33 was synthesised by PCR using primers5′-CAACCCAATTCTCTTCCCTCCTCCG-3′ and 5′-ACAAACACTITGGGCGGC-3′ and plasmidpA33 (a33 cDNA in pBluescript) as a template. The gene specific DNAfragments were radio labelled with alpha ³²P-ATP using the RadPrime kit(Life Technologies). Hybridisation was at 65° C. in 500 mM NaP-buffer,pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA. Filters were washed to a finalstringency of 0.1×SSC, 0.1% SDS at 65° C. (2×15 min) and finally exposedto X-omat AR (Kodak).

Transformation of Sugar Beet

Transgenic sugar beets (Beta vulgaris) were generated by a stomatalguard cell based transformation system (Hall et. al. 1996). Guard cellprotoplasts were obtained from shoot cultures of the diploid breedingline Bv-NF (Hall et. al. 1996). One million guard cell protoplasts weretransformed with 50 μg of the plasmid in the presence of PEG.Regeneration and selection of transformants was as described (Hall et.al. 1996). Plants were grown in a greenhouse at 18/15° C. (day/night)under a 16 h photoperiod. Two months old leaves were cut to preventspread with powdery mildew.

DNA and RNA Analysis of Transgenic Plants

Total DNA was isolated from young mature leaves which were harvested 3months after transfer of the plants to the greenhouse. DNA was digestedwith specific restriction enzymes. Total RNA was isolated from 2-5 monthold tap roots or tubers. After electrophoresis on 1% agarose, DNA or RNAwas blotted onto Hybond-N+(Amersham) and UV cross-linked. Filters werehybridised with a gene specific probe. The DNA fragment was radiolabelled with alpha ³²P-ATP using the RadPrime kit (Life Technologies).Hybridisation was at 65° C. in 500 mM NaP-buffer, pH 7.2, 7% SDS, 1%BSA, 1 mM EDTA. Filters were washed to a final stringency of 0.1×SSC,0.1% SDS at 65° C. (2×15 min) and finally exposed to X-omat AR (Kodak).

Enzyme Activity: Protein Extraction and Assay

Leaf and root tissues were ground in liquid nitrogen to a fine powder.Five hundred mg of the powdered tissue were extracted in 1 ml 50 mMBisTris pH 5.5, 1 mM EDTA, 1 mM MgSO_(4, 1) mM DTT, 1 mM PMSF, 20 mMNa-metabisulfite and 15 g 1⁻¹ PVPP. The extract was centrifuged (20 minat 10000×g) and the supernatant desalted and concentrated bycentrifugation through Centriprep 30 ultrafiltration devices (Amicon,Breda, The Netherlands). A 240 μl aliquot was mixed with 240 μl assaymixture, containing either 100 mM sucrose, GF₂ or GF₃ in 20 mM BisTrispH 5.5, 2 mM DTT and 0.01% Na-azide t(w/v). After 8, 16 and 24h ofincubation, an aliquot of the reaction mixture was boiled for 5 min andstore at −20° C. The assay mixtures were then analysed by HPAEC asdescribed below.

Analysis of Sugars and Inulin by TLC and HPAEC

The inulin composition of transgenic plants beet was measured by TLC(Thin Layer Chromatography) and HPAEC (High Performance Anion ExchangeChromatography). Two to five months after transfer of the plants to thegreenhouse, fresh plant material (500 mg) was collected and cut into 2mm thick slices with a sterile razor blade. A 20 mM phosphate buffer, pH7.0, of 100° C. was added to the slices to a final volume of 3 ml, andkept at 85° C. for 30 min, with occasional vortexing. The extract wascentrifuged at 14000 rpm and the supernatant collected. TLC analysis wasperformed on silica gel G (Schleicher and Schuell Nederland BV, TheNetherlands) developed two times in a mixture of1-butanol:2-propanol:water 3:12:4. Carbohydrates were stained with afructose-specific urea phosphoric spray (Wise et al., 1955). For HPAECanalysis, the extracts were deionized with a mixture of equal amounts ofQ-sepharose and S-sepharose Fast Flow (Pharmacia, Upssala, Sweden),which were pre-equilibrated in 20 mM phosphate buffer, pH 7.0. The ionexchanger was added to 50% of the total extract volume, mixed for 5 minat 600 rpm, then centrifuged at 14000 rpm for 2 min. The supernatant wasanalysed by High Pressure Anion Exchange Chromatography/PulsedAmperometric Detection (HPAEC-PAD, Dionex, The Netherlands) equippedwith 250×4 mm CarboPac PA1 anion exchange column and a 25×3 mm CarboPacPA guard column. Inulins were separated with a 80 min linear gradient of0 to 0.4 mol m⁻³ NaAc in 0.1 mol m⁻³ NaOH at a flow rate of 1 ml min⁻¹,or over 85 minutes with an aqueous gradient (A: NaOH 0.1 mol m⁻³; B:(0.1 mol m⁻³ NaOH+0.4 mol m⁻³ NaAc); C: NaOH 1 mol m⁻³) as follows: min0-5: A:B 96:4; min 5-15 linear gradient A:B from 96:4 to 60:40; min15-35: linear gradient A:B from 60:40 to 30:70; min 35-50 lineargradient A:B from 30:70 to 10:90; min 50-60 linear gradient A:B from10:90 to 0: 100; min 60-85 A:B 0:100; followed by regeneration min 0-5with A:B:C: 0:0:100 and min 5-30 with A:B 96:4, at a flow rate of 1 mlmin⁻¹. Standard grade inulin was used as a standard. CGC analysis wasmade according to the method described by L. De Leenheer et al.,Starch-Stärke, (1994), Vol. 46, p. 193-196.

EXAMPLES Example 1 Isolation of a New cDNA from the cDNA Library ofHelianthus tuberosus Homologous to sst103 and fft111 from Helianthustuberosus

An Uni-ZAP cDNA library, constructed from mRNA isolated from H.tuberosus tubers, was screened with a mixture of a 840 bp sst103 and a860 bp fft111 fragment. The sst103 specific fragment was obtained by PCRusing primers 5′-TGTCAGCCCATCCCTTGGAAAGG-3′ and5′-TACGCTGTCAACTCGTCGG-3′ and pSST103 (Van der Meer et al., 1998) astemplate. The fft111 specific fragment was obtained by PCR using5′-GTTCAACGCTGCTTGATCCACC-3′ and 5′-ACCACGGTCCTTCCAAACGG-3′ and pFFT111(Van der Meer et al, 1998) as template. Screening of about 100.000 cDNAclones yielded about 1200 positive clones, most of them most probablyrepresenting either a sst103 or a fft111 cDNA. Only the lambda ZAPclones giving a weak positive signal (84) were picked from the primaryscreening. After 34 rounds of purification, 11 clones were left. Fromthese clones, the pBluescript phagemids were excised from the uni-ZAPvector and the cloned insert characterised by restriction enzymeanalysis and sequencing. One clone with an about 2 kb insert, designateda33, was fully sequenced. The DNA sequence of a33 (Sequence ID. No.1)and the corresponding amino acid sequence (Sequence ID. No. 2) ispresented in FIG. 1. Sequence ID. No. 1, designated a33 has an openreading frame of 1845 base pairs and encodes a protein of 615 amino acidresidues (Sequence ID. No. 2). On DNA level, a33 shows a 70% identitywith the 1-SST encoding sst103 cDNA sequence from H. tuberosus and 54%identity with the 1-FFT encoding, fft111 cDNA sequence from H.tuberosus. At the amino acid level, a33 shows a 75% similarity withsst103 from H. tuberosus and a 58% similarity with fft111 from H.tuberosus.

Example 2 Isolation of a New cDNA from the cDNA Library of CichoriumintybusHomologous to sst103 and fft111 from Helianthus tuberosus

A Lambda TriplEx cDNA library, constructed from mRNA isolated from thetap roots of C. intybus, was screened with a mixture of a 840 bp sst103and a 860 bp fft111 fragment (see example 1). The primary screening ofabout 60.000 cDNA clones yielded about 700 positive clones, of which 96clones were picked. After 3-4 rounds of purification 81 clones wereleft. From 55 clones, the pTriplEx phagemids were excised from lambdaTrip1Ex vector and the cloned insert characterised by restriction enzymeanalysis and sequencing. The DNA sequence of one of the clones,designated c86b (Sequence ID. No. 3) and the corresponding amino acidsequence (Sequence ID. No. 4) is presented in FIG. 2A. Sequence ID. No.3, designated c86b, has an open reading frame of 1851 base pairs andencodes a protein of 617 amino acid residues (Sequence ID. No. 4). OnDNA level, c86b shows a 61% identity with sst103 from H. tuberosus and77% identity with fft111 from H. tuberosus. At the amino acid level,c86b shows a 53% similarity with sst103 from H. tuberosus and a 78%similarity with fft111 from H. tuberosus. The DNA sequence of anotherclone, designated c33 (Sequence ID. No. 5) and the corresponding amninoacid sequence (Sequence ID. No. 6) is presented in FIG. 2B. Sequence ID.No. 5 designated c33, has an open reading frame of 1920 base pairs andencodes a protein of 640 amino acid residues (Sequence ID. No. 6). OnDNA level, c33 shows a 97% identity with 1-SST from chicory (Genbankaccession no U81520). On amino acid level c33 shows a 88% /(similaritywith 1-SST from chicory (Genbank no U81520).

Example 3 Analysis of Genomic Organisation and Expression of a33 inHelianthus tuberosus

To estimate the number of a33 genes present in the Jerusalem artichokegenome, Southern blot analysis of digested H. tuberosus DNA wasperformed, using a radio labelled a33 fragment of 1140 bp as a probe.(Plant Journal 15, 489-500). Under stringent conditions (hybridisationat 65° C. and washing at 65° C., 0.1×SSC, 0.1% SDS), only one, maybe twofragments of the AflII digest hybridises to the a33 probe (FIG. 3),suggesting that only one, maybe two a33 genes are present in theJerusalem artichoke genome.

Transcript levels o a33, sst103 and fft111 were studied in differentorgans and different developmental stages of the tubers of Jerusalemartichoke (FIG. 4). In correspondence to earlier experiments (Van derMeer 1998), data in FIG. 4A shows that sst103 is highly expressed intubers, and to a less extent in fibrous roots, flowers and receptacle.The expression pattern of fft111 (FIG. 4B) resembles that of sst103,although in general the expression of fft111 is 3-10 times lower thanthat of sst103. In contrast to sst103 and fft111, a33 is not expressedto a measurable level in any of the tested tissues (FIG. 4C). Toquantify the a33, sst103 and fft111 expressing in tubers, 100.000plaques of the cDNA library of Jerusalem artichoke tubers were blottedonto Hybond filters, hybridised to either an a33, sst103 or fft111 probeat 65° C., then washed at high stringency (65° C., 0.1×SSC, 0.1% SDS).Expression levels of a33, sst103 or fft111 in tubers were 0.001, 1 and0.2%, respectively. If a33 encodes a functional fructosyltransferaseprotein, it does not effectively contribute to inulin synthesis intubers of Jerusalem artichoke. From a functional point of view, a33 canbe considered as a silent gene.

Example 4 Construction of a Chimeric a33 Gene Construct forTransformation into Potato

By PCR, using primers 5′-ATCAACATGTCTTCCACCCC-3′ and5′-TGGATCCTCAAGGCCGCCCTC-3′, an AflIII site was introduced at the firstATG (start of the open reading frame) and a BamHI site was introduceddown-stream of the stop codon of the full length a33 cDNA clone (pA33),isolated from Jerusalem artichoke. The newly obtained a33 PCR fragmentwas cloned into pMOSblue (Amersham Life Science) yielding pMOSA33.10.From the plasmid pFFT405 (see further, for a complete description),which contains the enhanced Cauliflower Mosaic Virus 35S promoter(enh35S), Alfalfa Mosaic Virus RNA4 leader sequence (amv), cDNA fft111from Jerusalem Artichoke and the nos terminator sequence (Tnos), thecomplete fft111 sequence was replaced by the a33 PCR fragment. Thecomplete a33 PCR fragment, resulting from a digestion of pMOSA33.10 withAflIII and BamHI (partial), was ligated into pFFT405, from which thefft111 was removed by a digestion with NcoI and BamHI (partial).Replacing the fft111 cDNA in pFFT405 with the a33 PCR fragment yieldedpA33.102. The enh35S-amv-a33-Tnos-fragment was cut from pA33.102 withSacI and SalI, and ligated into the plant transformation vector pBINPLUS(Van Engelen et al., 1995) digested with SacI/SalI, which resulted inpA33.236 (FIG. 5).

pFFT 405 was obtained by cloning the Penh35S-amv-fft111-Tnos-fragmentcut from pFFT209 (Van der Meer et al., 1998), with EcoRI (partialdigest) and HindIII into pBluescript SK+ (Stratagene, USA) cut withEcoRI/HindIII.

Example 5 Analysis of Transgenic Plants Expressing the a33 Gene

About 25 transgenic potato plants were generated harbouring the pA33.236construct. Ten potato plants were transformed with the Agrobacteriumstrain AGL0 lacking a binary vector. These plants were used as acontrol. Southern blot analysis of genomic DNA isolated from 22transformed plants showed that 17 plants has integrated into theirgenome less than 3 copies of the introduced chimeric gene (data notshown). Plant numbers 23, 27B, 74, 84 and 93 had integrated one copy.Northern analysis showed that a33 was highly expressed in plants numbers27B, 74 and 93, and less, but still clearly expressed in plant numbers23 and 84.

The carbohydrate composition of the a33 harbouring plants was analysedby two essentially different techniques: thin layer chromatography (TLC)and high pressure anion exchange chromatography (HPAEC). Analysis ofleaf extracts from the potato harbouring the pA33.26 construct showedthat plant numbers 23, 27B, 50, 83 and 93 accumulate a range of fructosecontaining compounds in leaves and tubers (FIG. 6). A comparison withthe inulin standard extracted from Jerusalem Artichoke, containinginulins up to a DP of 30, shows that the tuber extracts accumulateinulins up to a degree of polymerisation of at least 10 (FIG. 6),whereas the leaves accumulate inulins with a DP of 3, 4 and 5.

The presence of inulins in leaf extract and tuber extracts of thea33-harbouring plants is confirmed through HPAEC analysis. HPAECindicates that plant numbers 23, 27B, 93 (only 93 is shown in FIG. 7)accumulate inulins up to a degree of polymerisation of 11. This clearlyindicates that a33 encodes a fructosyltransferase enzyme, able tosynthesise inulins up to a DP of at least 11. Since the a33 encodes anenzyme that can catalyse all steps required for inulin synthesis,including reaction (1), the conversion of sucrose into GEF, the a33encoded enzyme (A33) belongs to the group of SST encoding genes.

Example 6 Construction of a Chimeric a33 Gene Construct forTransformation into Sugar

The complete chimeric a33 gene (Penh35S-amv-a33-Tnos) was cut frompA33.102 with NotI and SalI and ligated into pUCM2, digested withNotI/SalI, resulting in pUCA33.1. Plasmid pUCM2 is derived from thecloning vector pUCAP (Van Engelen et al. 1995).

To construct plasmid pUCM2 the multiple-cloning-site of plasmid pUCAPwas modified by the insertion of two adapters. First, adapter5′-TCGACCATATCGATGCATG-3′/5′-CATCGCTATTGG-3′, containing the restrictionsites SalI/ClaI/SphI, was cloned in the pUCAP plasmid digested withSahI/SphI. This resulted in plasmid pUCM1. Next, adapter5′-TAAGCGGCCGCAGATCTGG-3′/5′-AATTCCAGATCTGCGGCCGCTTAAT-3′, consisting ofPacI/NotI/BglII/EcoRI restriction sites, was cloned in plasmid pUCM1digested with PacI/EcoRI. This resulted in plasmid pUCM2. The completechimeric a33 gene (Penh35S-amv-a33-Tnos) was cut from pUCA33.1 with NotIand AscI and ligated into pUCPAT 34 digested with NotI/AscI, yieldingpUCPA33.342 (FIG. 8). pUCPA33.342 was used to transform guard cellprotoplasts of sugar beet.

Example 7 Construction of a Chimeric a33-c86b Gene for Transformationinto Sugar Beet

The complete chimeric C86B gene (Penh35S-amv-c86b-Tnos), which was cutfrom pC86B.2 with EcoRI and ClaI, and cloned into pUCPAT.34 (see example8), was digested with EcoR1 (partial digest) and ClaI. This resulted inpUCPC86B.342. The NotI/AscI fragment of pUCA33.1, containing the fulllength A33-cDNA (a33) was ligated into pUCPC86B.342 cut with NotI/AscI,yielding pUCPCA342.25. (FIG. 9). pUCPCA342.25 was used to transformguard cell protoplasts of sugar beet.

Example 8 Construction of a Chimeric sst103-fft111 Gene forTransformation into Sugar Beet

The pat gene, encoding phosphinotricin acetyl transferase (AgrEvo,Berlin, Germany), which confers bialaphos resistance, was cut frompIGPD7 (Hall et al., 1996) with EcoR1 and ligated into pUCM3, digestedwith EcoRI, which yielded pUCPAT34.

To construct plasmid pUCM3, plasmid pUCAP was digested with PacI/AscIand the whole multiple-cloning-site was replaced by adapter5′-TAAGGGGTACCACCATCGATACCGAATTCTACATGCATGCATGGAGATCTCCCAAGCTTCTAAGATGCGGCCGCTAAACATGG-3′/5′-CGCGCCATGTTTAGCGGCCGCATCTTAGAAGCTTGGGAGATCTCCATGCATGCATGTAGAATTCGGTATCGATGGTGGTACCCCTTAAT-3′The complete chimeric sst103 gene (Penh35S-amv-sst103-Tnos) was cut frompSST403 (Van der Meer et al., 1998) with EcoRI and ClaI and ligated intopUCM1, digested with EcoRI/ClaI, which resulted in pUCST21.

The complete chimeric fft111 gene (Penh35S-amv-fft111-Tnos) cut frompFFT405 with NotI and ClaI was ligated into pUCM2, cut with NotI/ClaI,resulting in pUCFT2l. The complete chimeric sst103 gene was cut frompUCST21 with PacI and ClaI and introduced into pUCPAT34, digested withPacI/ClaI, yielding pUCPS34.4. The complete chimeric fft111 gene was cutfrom pUCFT21 with NotI and AscI and ligated into pUCPS34.4, digestedwith NotI/AscI, yielding pUCPSF344.18 (FIG. 10). pUCPSF 344.18 was usedto transform guard cell protoplasts of sugar beet.

Example 9 Construction of a Chimeric sst103-c86b Gene for Transformationinto Sugar Beet

Using PCR and primer 5′-CCTCGAACCATGGAAACAGC-3′ an NcoI was introducedat the first ATG (start of the open reading frame) of the full lengthc86b cDNA clone. A BamHI site was introduced downstream of the stopcodon of c86b, using primer 5′-TAATAAAAGAGGATCCTCATGAAACG-3′. This c86bPCR fragment (1895 bp) was cloned into pCR-Script Amp SK(+) (Stratagene,USA) yielding p86BpCRscp. From the plasmid pFFT405, which contains theenhanced Cauliflower Mosaic Virus 35S promoter (Penh35S), Alfalfa MosaicVirus RNA4 leader sequence (amv), fft111 cDNA clone from JerusalemArtichoke and the nos terminator sequence (Tnos), the complete fft111sequence was replaced by the c86B PCR fragment. The c86B fragment, cutfrom p86BpCRscp with NcoI and BamHI, was ligated into pFFT405 cut withNcoI and BamHI, yielding pC86B2. The NotI/ClaI fragment of pC86B2(enh35S-amv-sst103-Tnos) was cloned into pUCM2 cut with NotI/ClaI,yielding pUC86B2. The NotI/AscI fragment of pUC86B2 was cloned into thepUCPS344 digested with NotI/AscI, resulting in pUCPSC344.25 (FIG. 11).pUCPSC344.25 was used to transform guard cell protoplasts of sugar beet.

Example 10 Construction of a Chimeric a33-fft111 Gene for Transformationinto Sugar Beet

From the plasmid pUCPA33.342, the complete recombinant a33 gene(Penh35S-amv-sst103-Tnos) and the complete pat cassette were excisedwith ClaI, then introduced into pUCFT21 cut with ClaI, yieldingpUCPAF21.17 (FIG. 12)

Example 11 Analysis of Sugar Beet plants Comprising the pUCPSF344.18Plasmid

Two fructosyltransferase encoding cDNAs from H. tuberosus (sst 103 andfft111), and the pat gene conferring bialaphos resistance were clonedinto the pUCM3 vector. The resulting plasmid pUCPSF344.18 (FIG. 10) wasused to transform stomatal guard cell protoplasts of sugar beet.

Bialophos-resistent calli were obtained after bialophos selection.Regeneration and selection of transformants was as described (Hall etal. 1996). Transgenic sugar beets were analyzed by Northern analysis(FIG. 13): as the sst103 probe a DNA fragment was used which wasprepared by PCR using primers 5′-TTGAATAGAACATCGGGCTCTAGCG-3′ and5′-TACGCTGTCAACTCGTCGG-3′ and plasmid pUCFT21 (example 8) as a template;as the fft111 probe a DNA fragment was used which was prepared by PCRusing primers 5′-GTTCAACGCTGCTGGATCCACC-3′ and5′-ACCACGGTCCTTCCAAACGG-3′ and plasmid pUCFT21 (example 8) as atemplate. Line 7PSF22 expressed both the sst103 and the fft111 gene(FIG. 13, lane 1). This line was further analyzed by HPAEC. The inulinprofile can be described as a mixture of inulin molecules up to a DP of9 (FIG. 14). The inulin profile of sugar beet line 7PSF22 is essentiallydifferent from the inulin profile of transgenic sugar beet linescomprising only the sst103 gene (Sevenier et al. 1998). The sugar beetcomprising only the sst103 gene accumulates inulins up to a DP of 5(GF₂, GF₃ and GF₄; Sevenier et al. 1998). The inulin profile of line7PSF22 is also different from the standard grade inulin from chicory(FIG. 15), which comprises of a mixture of inulins up to a DP of about55.

Example 12 Analysis of Sugar Beet Plants Comprising the pUCPSC 344.14Plasmid

The plasmid prepared according to the description in example 9, was usedto transform stomatal guard cell protoplasts of sugar beet.Bialophos-resistent calli were obtained after bialophos selection.Regeneration and selection of transformants was as described (Hall etal. 1996). Transgenic sugar beets were analysed by Northern analysis(FIG. 13): as a c86b probe a DNA fragment was used, which was preparedby PCR using primers 5′-GTGACCTTGAGGATGCATCC-3′ and5′-TCGGTTGCACCCGCGCTCG-3′ and plasmid pUC86B2 (example 9) as a template.

Line 9PSC2, expressing both the sst103 and the c86b gene (FIG. 13, lane2), was selected for further analysis by HPAEC. The inulin profile ofsugar beet line 9PSC2 can be described as a polydisperse mixture ofinulin molecules with a DP ranging from 3 to 15 (FIG. 16). The inulinprofile of sugar beet line 9PSC2 is essentially different fromtransgenic sugar beet lines comprising only the sst103 gene (Sevenier etal. 1998), but also from line 7PSF22 comprising a combination of sst103and fft111, which accumulate inulin molecules up to a DP of 9 (FIG. 14).The inulin profile of line 9PSC2 is also different from the standardgrade inulin from chicory (FIG. 15), which comprises of inulins up to aDP of about 55.

Example 13 Analysis of Sugar Beet Plants Comprising the pUCPAF21.17Plasmid

The plasmid prepared according to the description in example 10, wasused to transform stomatal guard cell protoplasts of sugar beet.Bialophos-resistent calli were obtained after bialophos selection.Regeneration and selection of transformants was as described (Hall etal. 1996). Transgenic sugar beets were analysed by Northern analysis: asthe a33 probe a DNA fragment was used, which was prepared by PCR usingprimers 5′-CAACCCAATTCTCTTCCCTCCTCCG-3′ and 5′-ACAAACACTTTCGGCCGCC-3′and plasmid pUCA33.1 (example 6) as a template. Lines 8PAF1 and 8PAF5which were expressing both the a33 and the fft111 gene (FIG. 13, lanes 3and 4, respectively), were analysed by HPAEC. Sugar beet line 8PAF5accumulates a inuline molecules ranging from DP 3-22 (FIG. 17). Sugarbeet line 8PAF1 accumulates a mixture of inulin molecules with a DPranging from 3 to 26 (FIG. 18). In contrast to lines 7PSF22 (example11), 9PSC2 (example 12) and 8PAF5 (example 13), in which GF₂ isinvariably the most abundant inulin molecule, in line 8PAF1, theconcentrations of GF₂, GF₃, GF₄ and GF₅ are in the same range, being2.5. 2.5. 2.4 and 2.2% of total dry matter, respectively. Surprisingly,line 8PAF1 also accumulates significant amounts of F₂, F₃, F₄, F₅, F₆,F₇, F₈ and F₉, being 0.4, 0.4, 0.5, 0.4, 0.4, 0.3, 0.2, 0.1% of totaldry weight, respectively (CGC-analysis results).

The inulin profiles of sugar beet lines 8PAF1 and 8PAF5 are essentiallydifferent from from sugar line 7PSF22 comprising a combination of sst103and fft11, which accumulate inuline molecules up to a DP of only 9. Theinulin profile of lines 8PAF1 and 8PAF5 are also different from thestandard grade inulin from chicory (FIG. 15), which comprises of inulinsup to a DP of about 55.

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6 1 2008 DNA Helianthus tuberosus CDS (13)..(1857) 1 gcaaaaatca cc atggct tcc acc ccc acc acc cct ctt att act cac aat 51 Met Ala Ser Thr ProThr Thr Pro Leu Ile Thr His Asn 1 5 10 gac ctt gaa caa cgc ccg gaa tcgacc gag tct cca ccc ggt cga tca 99 Asp Leu Glu Gln Arg Pro Glu Ser ThrGlu Ser Pro Pro Gly Arg Ser 15 20 25 tcc atc gta aag atc ctc act gga ttattt gtg tcc att ctt gtt ctt 147 Ser Ile Val Lys Ile Leu Thr Gly Leu PheVal Ser Ile Leu Val Leu 30 35 40 45 tca tca ttg gct gca ata aca cac cggaaa act ccc ttg cag tcc acc 195 Ser Ser Leu Ala Ala Ile Thr His Arg LysThr Pro Leu Gln Ser Thr 50 55 60 aca gtt gat att gaa cca tcg aca agc agtccg aag gag gtt gtg gga 243 Thr Val Asp Ile Glu Pro Ser Thr Ser Ser ProLys Glu Val Val Gly 65 70 75 gcg gat gat agc att gaa tgg caa cga tct gcttac cat ttt caa ccc 291 Ala Asp Asp Ser Ile Glu Trp Gln Arg Ser Ala TyrHis Phe Gln Pro 80 85 90 gat aaa aat ttc att agc gat cct gat ggt cca ctgtat tac aag gga 339 Asp Lys Asn Phe Ile Ser Asp Pro Asp Gly Pro Leu TyrTyr Lys Gly 95 100 105 tgg tac cac tta ttc tac caa tac aat ccg ggg tcagcc att tgg ggc 387 Trp Tyr His Leu Phe Tyr Gln Tyr Asn Pro Gly Ser AlaIle Trp Gly 110 115 120 125 aac ata aca tgg ggt cat gca gtc tcg aaa gacctc atc aat tgg ttc 435 Asn Ile Thr Trp Gly His Ala Val Ser Lys Asp LeuIle Asn Trp Phe 130 135 140 cac ctc cct tta gcc atg gtt ccg gat cac tggtac gac atc cat ggt 483 His Leu Pro Leu Ala Met Val Pro Asp His Trp TyrAsp Ile His Gly 145 150 155 gtc atg act ggg tcc gcc acc atc ctc ccc aatggc caa atc ttc atg 531 Val Met Thr Gly Ser Ala Thr Ile Leu Pro Asn GlyGln Ile Phe Met 160 165 170 ctt tat agc ggc aac gcc tac gac ctc tct cagctt caa tgc ctc gcg 579 Leu Tyr Ser Gly Asn Ala Tyr Asp Leu Ser Gln LeuGln Cys Leu Ala 175 180 185 tac ccc aaa aat gct tct gat cca ctt ctt atcgaa tgg gtc aaa tac 627 Tyr Pro Lys Asn Ala Ser Asp Pro Leu Leu Ile GluTrp Val Lys Tyr 190 195 200 205 gaa ggc aac cca att ctc ttc cct cct ccgggc gtg ggt ctc aaa gac 675 Glu Gly Asn Pro Ile Leu Phe Pro Pro Pro GlyVal Gly Leu Lys Asp 210 215 220 ttt agg gac ccg tca tct ctt tgg att gggccc gat ggg aag tac cga 723 Phe Arg Asp Pro Ser Ser Leu Trp Ile Gly ProAsp Gly Lys Tyr Arg 225 230 235 atg gtt atg ggc tcc aag cac aat aat acaatt ggt tgt gct tta att 771 Met Val Met Gly Ser Lys His Asn Asn Thr IleGly Cys Ala Leu Ile 240 245 250 tac cac acc act aat ttc acc cat ttt gaattg ttg gat gag gtg ctc 819 Tyr His Thr Thr Asn Phe Thr His Phe Glu LeuLeu Asp Glu Val Leu 255 260 265 cat tcg gtt cag ggt acg ggt atg tgg gaatgt gtt gat ctt tac ccc 867 His Ser Val Gln Gly Thr Gly Met Trp Glu CysVal Asp Leu Tyr Pro 270 275 280 285 gta tcc acg acc gag aca aac ggg ttggat atg tcg aat cat gag tcg 915 Val Ser Thr Thr Glu Thr Asn Gly Leu AspMet Ser Asn His Glu Ser 290 295 300 ggt gct aag tat gtg ttg aag caa agtggg gat gag gat aga cat gat 963 Gly Ala Lys Tyr Val Leu Lys Gln Ser GlyAsp Glu Asp Arg His Asp 305 310 315 tgg tat gca att ggg gca tat gac gtggtt cat gat aaa tgg tat ccg 1011 Trp Tyr Ala Ile Gly Ala Tyr Asp Val ValHis Asp Lys Trp Tyr Pro 320 325 330 gat gat ccg gaa atg gat ttg ggt atcggg ttg aga tat gat tat gga 1059 Asp Asp Pro Glu Met Asp Leu Gly Ile GlyLeu Arg Tyr Asp Tyr Gly 335 340 345 aag ttt tat gct tca aag acg ttt tatgac ccg agt aag aag agg cgg 1107 Lys Phe Tyr Ala Ser Lys Thr Phe Tyr AspPro Ser Lys Lys Arg Arg 350 355 360 365 gtc tta tgg ggc tat gtt ggt gaaacg gat cct caa aaa gat gac ctc 1155 Val Leu Trp Gly Tyr Val Gly Glu ThrAsp Pro Gln Lys Asp Asp Leu 370 375 380 gag aaa gga tgg gcc aat att ttgaat gtt cct aga acc gtg gtg ttg 1203 Glu Lys Gly Trp Ala Asn Ile Leu AsnVal Pro Arg Thr Val Val Leu 385 390 395 gac acg aag acg caa agt aac ttgatt caa tgg ccg gtc gag gaa aca 1251 Asp Thr Lys Thr Gln Ser Asn Leu IleGln Trp Pro Val Glu Glu Thr 400 405 410 gaa act ttg aga tct gaa gag tacgat gag ttc aaa gat gtt gag ttg 1299 Glu Thr Leu Arg Ser Glu Glu Tyr AspGlu Phe Lys Asp Val Glu Leu 415 420 425 cgg cct gga tca ctt gtc ccg cttgat ata ggc tca gcc aca cag ttg 1347 Arg Pro Gly Ser Leu Val Pro Leu AspIle Gly Ser Ala Thr Gln Leu 430 435 440 445 gac ata agt gcc tca ttc gaggtt gat gaa gct ttg ctg ggt gca acc 1395 Asp Ile Ser Ala Ser Phe Glu ValAsp Glu Ala Leu Leu Gly Ala Thr 450 455 460 tta gaa gcc gat gtg ttg ttcaac tgc acc acg agc gag ggt tca gcc 1443 Leu Glu Ala Asp Val Leu Phe AsnCys Thr Thr Ser Glu Gly Ser Ala 465 470 475 atg agg ggt gtt ttg gga ccgttt ggg ctt gtg gtt ctt gca gat tcg 1491 Met Arg Gly Val Leu Gly Pro PheGly Leu Val Val Leu Ala Asp Ser 480 485 490 gca ctt tca gaa caa act cctgtt tac ttc tac att gcg aaa aac ttg 1539 Ala Leu Ser Glu Gln Thr Pro ValTyr Phe Tyr Ile Ala Lys Asn Leu 495 500 505 gat ggc act tca aga act tatttc tgt gct gat gaa tca aga tca tca 1587 Asp Gly Thr Ser Arg Thr Tyr PheCys Ala Asp Glu Ser Arg Ser Ser 510 515 520 525 aag ctt tta gat gtg ggcaag atg gta tat gga agc agt gtt cct gta 1635 Lys Leu Leu Asp Val Gly LysMet Val Tyr Gly Ser Ser Val Pro Val 530 535 540 ctc cat ggg gaa aac tacgac atg agg tta ttg gtg gat cat tca ata 1683 Leu His Gly Glu Asn Tyr AspMet Arg Leu Leu Val Asp His Ser Ile 545 550 555 gtc gaa agc ttt gca caagga gga aga acg gtg att aca tca aga gtg 1731 Val Glu Ser Phe Ala Gln GlyGly Arg Thr Val Ile Thr Ser Arg Val 560 565 570 tat cct aca atg gca atctat gat gcc gcc aaa gtg ttt gtg ttc aac 1779 Tyr Pro Thr Met Ala Ile TyrAsp Ala Ala Lys Val Phe Val Phe Asn 575 580 585 aat gca act gga atc actgtt aag gca tct ctc aag att tgg aag atg 1827 Asn Ala Thr Gly Ile Thr ValLys Ala Ser Leu Lys Ile Trp Lys Met 590 595 600 605 ggt gga gca caa ctcaac cct ttt cct ttc taattagttt agttggcttc 1877 Gly Gly Ala Gln Leu AsnPro Phe Pro Phe 610 615 attagttggt gacgttttgg tgaatttgta agcttgttgtagtgagggcg gccttgatga 1937 ttaatattgc cattgtaaaa cttccatttt tttaaaaaaataatcgattt aaaagttttt 1997 ttaaaaaaaa a 2008 2 615 PRT Helianthustuberosus 2 Met Ala Ser Thr Pro Thr Thr Pro Leu Ile Thr His Asn Asp LeuGlu 1 5 10 15 Gln Arg Pro Glu Ser Thr Glu Ser Pro Pro Gly Arg Ser SerIle Val 20 25 30 Lys Ile Leu Thr Gly Leu Phe Val Ser Ile Leu Val Leu SerSer Leu 35 40 45 Ala Ala Ile Thr His Arg Lys Thr Pro Leu Gln Ser Thr ThrVal Asp 50 55 60 Ile Glu Pro Ser Thr Ser Ser Pro Lys Glu Val Val Gly AlaAsp Asp 65 70 75 80 Ser Ile Glu Trp Gln Arg Ser Ala Tyr His Phe Gln ProAsp Lys Asn 85 90 95 Phe Ile Ser Asp Pro Asp Gly Pro Leu Tyr Tyr Lys GlyTrp Tyr His 100 105 110 Leu Phe Tyr Gln Tyr Asn Pro Gly Ser Ala Ile TrpGly Asn Ile Thr 115 120 125 Trp Gly His Ala Val Ser Lys Asp Leu Ile AsnTrp Phe His Leu Pro 130 135 140 Leu Ala Met Val Pro Asp His Trp Tyr AspIle His Gly Val Met Thr 145 150 155 160 Gly Ser Ala Thr Ile Leu Pro AsnGly Gln Ile Phe Met Leu Tyr Ser 165 170 175 Gly Asn Ala Tyr Asp Leu SerGln Leu Gln Cys Leu Ala Tyr Pro Lys 180 185 190 Asn Ala Ser Asp Pro LeuLeu Ile Glu Trp Val Lys Tyr Glu Gly Asn 195 200 205 Pro Ile Leu Phe ProPro Pro Gly Val Gly Leu Lys Asp Phe Arg Asp 210 215 220 Pro Ser Ser LeuTrp Ile Gly Pro Asp Gly Lys Tyr Arg Met Val Met 225 230 235 240 Gly SerLys His Asn Asn Thr Ile Gly Cys Ala Leu Ile Tyr His Thr 245 250 255 ThrAsn Phe Thr His Phe Glu Leu Leu Asp Glu Val Leu His Ser Val 260 265 270Gln Gly Thr Gly Met Trp Glu Cys Val Asp Leu Tyr Pro Val Ser Thr 275 280285 Thr Glu Thr Asn Gly Leu Asp Met Ser Asn His Glu Ser Gly Ala Lys 290295 300 Tyr Val Leu Lys Gln Ser Gly Asp Glu Asp Arg His Asp Trp Tyr Ala305 310 315 320 Ile Gly Ala Tyr Asp Val Val His Asp Lys Trp Tyr Pro AspAsp Pro 325 330 335 Glu Met Asp Leu Gly Ile Gly Leu Arg Tyr Asp Tyr GlyLys Phe Tyr 340 345 350 Ala Ser Lys Thr Phe Tyr Asp Pro Ser Lys Lys ArgArg Val Leu Trp 355 360 365 Gly Tyr Val Gly Glu Thr Asp Pro Gln Lys AspAsp Leu Glu Lys Gly 370 375 380 Trp Ala Asn Ile Leu Asn Val Pro Arg ThrVal Val Leu Asp Thr Lys 385 390 395 400 Thr Gln Ser Asn Leu Ile Gln TrpPro Val Glu Glu Thr Glu Thr Leu 405 410 415 Arg Ser Glu Glu Tyr Asp GluPhe Lys Asp Val Glu Leu Arg Pro Gly 420 425 430 Ser Leu Val Pro Leu AspIle Gly Ser Ala Thr Gln Leu Asp Ile Ser 435 440 445 Ala Ser Phe Glu ValAsp Glu Ala Leu Leu Gly Ala Thr Leu Glu Ala 450 455 460 Asp Val Leu PheAsn Cys Thr Thr Ser Glu Gly Ser Ala Met Arg Gly 465 470 475 480 Val LeuGly Pro Phe Gly Leu Val Val Leu Ala Asp Ser Ala Leu Ser 485 490 495 GluGln Thr Pro Val Tyr Phe Tyr Ile Ala Lys Asn Leu Asp Gly Thr 500 505 510Ser Arg Thr Tyr Phe Cys Ala Asp Glu Ser Arg Ser Ser Lys Leu Leu 515 520525 Asp Val Gly Lys Met Val Tyr Gly Ser Ser Val Pro Val Leu His Gly 530535 540 Glu Asn Tyr Asp Met Arg Leu Leu Val Asp His Ser Ile Val Glu Ser545 550 555 560 Phe Ala Gln Gly Gly Arg Thr Val Ile Thr Ser Arg Val TyrPro Thr 565 570 575 Met Ala Ile Tyr Asp Ala Ala Lys Val Phe Val Phe AsnAsn Ala Thr 580 585 590 Gly Ile Thr Val Lys Ala Ser Leu Lys Ile Trp LysMet Gly Gly Ala 595 600 605 Gln Leu Asn Pro Phe Pro Phe 610 615 3 1982DNA Cichorium intybus CDS (40)..(1890) 3 tcgcggccgc gtcgacacttggcccatttc cctcgaaca atg aaa aca gcc gaa 54 Met Lys Thr Ala Glu 1 5 ccctta agt gac ctt gag gat gca tcc aac cgc act ccc cta cta gac 102 Pro LeuSer Asp Leu Glu Asp Ala Ser Asn Arg Thr Pro Leu Leu Asp 10 15 20 cac cctgca cca cca ccg gcc gcc gtg aaa aag cag tcg ttc gtc agg 150 His Pro AlaPro Pro Pro Ala Ala Val Lys Lys Gln Ser Phe Val Arg 25 30 35 gtt ctg tccagt atc act ttg gtg tct ctg ttc ttc gtt tta gct ttc 198 Val Leu Ser SerIle Thr Leu Val Ser Leu Phe Phe Val Leu Ala Phe 40 45 50 gta ctc atc gtcctg aac cag caa gat tcc acg aac gcc act gcc aat 246 Val Leu Ile Val LeuAsn Gln Gln Asp Ser Thr Asn Ala Thr Ala Asn 55 60 65 tta gca ctg ccg gagaaa tct tcg gct caa cac tat cag tcc gat cgc 294 Leu Ala Leu Pro Glu LysSer Ser Ala Gln His Tyr Gln Ser Asp Arg 70 75 80 85 ctg aca tgg gaa agaaca gct tac cat ttt cag cca gcc aaa aat ttc 342 Leu Thr Trp Glu Arg ThrAla Tyr His Phe Gln Pro Ala Lys Asn Phe 90 95 100 atc tac gat ccc aatggg cca ctg ttc cac atg ggt tgg tac cat ctt 390 Ile Tyr Asp Pro Asn GlyPro Leu Phe His Met Gly Trp Tyr His Leu 105 110 115 ttc tat caa tac aacccg tac gct cca att tgg ggc aac atg tca tgg 438 Phe Tyr Gln Tyr Asn ProTyr Ala Pro Ile Trp Gly Asn Met Ser Trp 120 125 130 ggt cac gcc gtg tccaaa gac atg atc aac tgg ttc gag ctt ccc gta 486 Gly His Ala Val Ser LysAsp Met Ile Asn Trp Phe Glu Leu Pro Val 135 140 145 gcc ttg aca cca accgag tgg tac gat atc gag ggc gtc tta tcc ggg 534 Ala Leu Thr Pro Thr GluTrp Tyr Asp Ile Glu Gly Val Leu Ser Gly 150 155 160 165 tcc acc acg gccctc ccc aac ggt caa atc ttt gca ttg tac acc gga 582 Ser Thr Thr Ala LeuPro Asn Gly Gln Ile Phe Ala Leu Tyr Thr Gly 170 175 180 aat gct aat gatttc tct caa cta caa tgc aaa gct gtt ccg tta aac 630 Asn Ala Asn Asp PheSer Gln Leu Gln Cys Lys Ala Val Pro Leu Asn 185 190 195 aca tct gac ccactc ctt ctc gag tgg gtc aaa tac gag aat aac cca 678 Thr Ser Asp Pro LeuLeu Leu Glu Trp Val Lys Tyr Glu Asn Asn Pro 200 205 210 atc ttg ttc actcca cca ggg att gga tta aaa gac tat cgg gac ccg 726 Ile Leu Phe Thr ProPro Gly Ile Gly Leu Lys Asp Tyr Arg Asp Pro 215 220 225 tct aca gtt tggacg ggt cct gat gga aaa cat cgg atg atc atg ggc 774 Ser Thr Val Trp ThrGly Pro Asp Gly Lys His Arg Met Ile Met Gly 230 235 240 245 act aaa ataaat cgt act gga ctc gta ctt gtt tac cat act acc gac 822 Thr Lys Ile AsnArg Thr Gly Leu Val Leu Val Tyr His Thr Thr Asp 250 255 260 ttc aca aactat gta atg ttg gag gag ccg ttg cat tcg gtt ccc gat 870 Phe Thr Asn TyrVal Met Leu Glu Glu Pro Leu His Ser Val Pro Asp 265 270 275 acc gat atgtgg gaa tgt gtt gac ttg tac cct gtg tca aca att aat 918 Thr Asp Met TrpGlu Cys Val Asp Leu Tyr Pro Val Ser Thr Ile Asn 280 285 290 gac agc gcactt gat atc gcg gct tat ggt ccc gat atg aag cat gtg 966 Asp Ser Ala LeuAsp Ile Ala Ala Tyr Gly Pro Asp Met Lys His Val 295 300 305 att aaa gaaagt tgg gag gga cat ggg atg gac tgg tac tcg att ggg 1014 Ile Lys Glu SerTrp Glu Gly His Gly Met Asp Trp Tyr Ser Ile Gly 310 315 320 325 aca tatgat gtg ata aac gat aag tgg acc ccg gat aac ccg gaa ttg 1062 Thr Tyr AspVal Ile Asn Asp Lys Trp Thr Pro Asp Asn Pro Glu Leu 330 335 340 gac gtgggt att ggg tta aga gtc gat tac ggg agg ttt ttt gca tca 1110 Asp Val GlyIle Gly Leu Arg Val Asp Tyr Gly Arg Phe Phe Ala Ser 345 350 355 aag agtctt tat gac ccg ttg aag aaa cgg agg gtc act tgg ggt tat 1158 Lys Ser LeuTyr Asp Pro Leu Lys Lys Arg Arg Val Thr Trp Gly Tyr 360 365 370 gtt gcagaa tcg gac agt gcg gac cag gac ctt aat aga ggg tgg gct 1206 Val Ala GluSer Asp Ser Ala Asp Gln Asp Leu Asn Arg Gly Trp Ala 375 380 385 act atttac aac gtt gca aga acc att gtg cta gat aga aag acc gga 1254 Thr Ile TyrAsn Val Ala Arg Thr Ile Val Leu Asp Arg Lys Thr Gly 390 395 400 405 acccat cta ctt cat tgg cct gtt gag gaa att gag agt ttg aga tat 1302 Thr HisLeu Leu His Trp Pro Val Glu Glu Ile Glu Ser Leu Arg Tyr 410 415 420 gatggt cgt gaa ttt aaa gag atc gag ctt gca ccg ggt tcg atc atg 1350 Asp GlyArg Glu Phe Lys Glu Ile Glu Leu Ala Pro Gly Ser Ile Met 425 430 435 ccactc gac ata ggc ccg gct acg cag ttg gac ata gtt gcc aca ttt 1398 Pro LeuAsp Ile Gly Pro Ala Thr Gln Leu Asp Ile Val Ala Thr Phe 440 445 450 gaggtg gaa caa gag acg ttt atg agg aca agt gac aca aat ggt gaa 1446 Glu ValGlu Gln Glu Thr Phe Met Arg Thr Ser Asp Thr Asn Gly Glu 455 460 465 tacggt tgc acc acg agc gcg ggt gca acc gaa agg gga agt ttg gga 1494 Tyr GlyCys Thr Thr Ser Ala Gly Ala Thr Glu Arg Gly Ser Leu Gly 470 475 480 485ccg ttt ggg atc gcg gtt ctt gct gat gga aca ctc tcg gaa tta act 1542 ProPhe Gly Ile Ala Val Leu Ala Asp Gly Thr Leu Ser Glu Leu Thr 490 495 500cct gtg tat ttc tat att tct aaa aag aca gat gga agc gtt gca aca 1590 ProVal Tyr Phe Tyr Ile Ser Lys Lys Thr Asp Gly Ser Val Ala Thr 505 510 515cat ttt tgt acc gat aag cta agg tca tca ctg gat tat gac ggg gag 1638 HisPhe Cys Thr Asp Lys Leu Arg Ser Ser Leu Asp Tyr Asp Gly Glu 520 525 530aga gtg gta tac ggg agc act gtc cct gta ctc gat ggt gaa gaa ctc 1686 ArgVal Val Tyr Gly Ser Thr Val Pro Val Leu Asp Gly Glu Glu Leu 535 540 545aca atg agg tta ctg gtg gat cat tca gta gtg gag ggg ttt gca atg 1734 ThrMet Arg Leu Leu Val Asp His Ser Val Val Glu Gly Phe Ala Met 550 555 560565 gga gga agg aca gta atg aca tca cga gtg tat ccc aca aag gca ata 1782Gly Gly Arg Thr Val Met Thr Ser Arg Val Tyr Pro Thr Lys Ala Ile 570 575580 tat gaa gga gcc aag atc ttc ttg ttc aac aat gcg act cat acc agt 1830Tyr Glu Gly Ala Lys Ile Phe Leu Phe Asn Asn Ala Thr His Thr Ser 585 590595 gtg aag gca tct ctc aag atc tgg caa ata gct tct gta cga atc cag 1878Val Lys Ala Ser Leu Lys Ile Trp Gln Ile Ala Ser Val Arg Ile Gln 600 605610 cct tac cct ttt tagttatttc gtttcatgaa catgctcttt tattatatat 1930 ProTyr Pro Phe 615 attcatgtat tttattttcc ttctaggtaa aaaaaaaaaa aaaaaaaaaaaa 1982 4 617 PRT Cichorium intybus 4 Met Lys Thr Ala Glu Pro Leu SerAsp Leu Glu Asp Ala Ser Asn Arg 1 5 10 15 Thr Pro Leu Leu Asp His ProAla Pro Pro Pro Ala Ala Val Lys Lys 20 25 30 Gln Ser Phe Val Arg Val LeuSer Ser Ile Thr Leu Val Ser Leu Phe 35 40 45 Phe Val Leu Ala Phe Val LeuIle Val Leu Asn Gln Gln Asp Ser Thr 50 55 60 Asn Ala Thr Ala Asn Leu AlaLeu Pro Glu Lys Ser Ser Ala Gln His 65 70 75 80 Tyr Gln Ser Asp Arg LeuThr Trp Glu Arg Thr Ala Tyr His Phe Gln 85 90 95 Pro Ala Lys Asn Phe IleTyr Asp Pro Asn Gly Pro Leu Phe His Met 100 105 110 Gly Trp Tyr His LeuPhe Tyr Gln Tyr Asn Pro Tyr Ala Pro Ile Trp 115 120 125 Gly Asn Met SerTrp Gly His Ala Val Ser Lys Asp Met Ile Asn Trp 130 135 140 Phe Glu LeuPro Val Ala Leu Thr Pro Thr Glu Trp Tyr Asp Ile Glu 145 150 155 160 GlyVal Leu Ser Gly Ser Thr Thr Ala Leu Pro Asn Gly Gln Ile Phe 165 170 175Ala Leu Tyr Thr Gly Asn Ala Asn Asp Phe Ser Gln Leu Gln Cys Lys 180 185190 Ala Val Pro Leu Asn Thr Ser Asp Pro Leu Leu Leu Glu Trp Val Lys 195200 205 Tyr Glu Asn Asn Pro Ile Leu Phe Thr Pro Pro Gly Ile Gly Leu Lys210 215 220 Asp Tyr Arg Asp Pro Ser Thr Val Trp Thr Gly Pro Asp Gly LysHis 225 230 235 240 Arg Met Ile Met Gly Thr Lys Ile Asn Arg Thr Gly LeuVal Leu Val 245 250 255 Tyr His Thr Thr Asp Phe Thr Asn Tyr Val Met LeuGlu Glu Pro Leu 260 265 270 His Ser Val Pro Asp Thr Asp Met Trp Glu CysVal Asp Leu Tyr Pro 275 280 285 Val Ser Thr Ile Asn Asp Ser Ala Leu AspIle Ala Ala Tyr Gly Pro 290 295 300 Asp Met Lys His Val Ile Lys Glu SerTrp Glu Gly His Gly Met Asp 305 310 315 320 Trp Tyr Ser Ile Gly Thr TyrAsp Val Ile Asn Asp Lys Trp Thr Pro 325 330 335 Asp Asn Pro Glu Leu AspVal Gly Ile Gly Leu Arg Val Asp Tyr Gly 340 345 350 Arg Phe Phe Ala SerLys Ser Leu Tyr Asp Pro Leu Lys Lys Arg Arg 355 360 365 Val Thr Trp GlyTyr Val Ala Glu Ser Asp Ser Ala Asp Gln Asp Leu 370 375 380 Asn Arg GlyTrp Ala Thr Ile Tyr Asn Val Ala Arg Thr Ile Val Leu 385 390 395 400 AspArg Lys Thr Gly Thr His Leu Leu His Trp Pro Val Glu Glu Ile 405 410 415Glu Ser Leu Arg Tyr Asp Gly Arg Glu Phe Lys Glu Ile Glu Leu Ala 420 425430 Pro Gly Ser Ile Met Pro Leu Asp Ile Gly Pro Ala Thr Gln Leu Asp 435440 445 Ile Val Ala Thr Phe Glu Val Glu Gln Glu Thr Phe Met Arg Thr Ser450 455 460 Asp Thr Asn Gly Glu Tyr Gly Cys Thr Thr Ser Ala Gly Ala ThrGlu 465 470 475 480 Arg Gly Ser Leu Gly Pro Phe Gly Ile Ala Val Leu AlaAsp Gly Thr 485 490 495 Leu Ser Glu Leu Thr Pro Val Tyr Phe Tyr Ile SerLys Lys Thr Asp 500 505 510 Gly Ser Val Ala Thr His Phe Cys Thr Asp LysLeu Arg Ser Ser Leu 515 520 525 Asp Tyr Asp Gly Glu Arg Val Val Tyr GlySer Thr Val Pro Val Leu 530 535 540 Asp Gly Glu Glu Leu Thr Met Arg LeuLeu Val Asp His Ser Val Val 545 550 555 560 Glu Gly Phe Ala Met Gly GlyArg Thr Val Met Thr Ser Arg Val Tyr 565 570 575 Pro Thr Lys Ala Ile TyrGlu Gly Ala Lys Ile Phe Leu Phe Asn Asn 580 585 590 Ala Thr His Thr SerVal Lys Ala Ser Leu Lys Ile Trp Gln Ile Ala 595 600 605 Ser Val Arg IleGln Pro Tyr Pro Phe 610 615 5 2157 DNA Cichorium intybus CDS(22)..(1941) 5 cgcggccgcg tcgaccccca c atg gct tcc tct acc acc gcc accacc cct 51 Met Ala Ser Ser Thr Thr Ala Thr Thr Pro 1 5 10 ctc atc ctccgt gat gag act caa atc agc cca caa cta gct gga tct 99 Leu Ile Leu ArgAsp Glu Thr Gln Ile Ser Pro Gln Leu Ala Gly Ser 15 20 25 ccg gtg ggt cggcgt tta tcc atg gcc aat atc ctt tcc ggg atc ctc 147 Pro Val Gly Arg ArgLeu Ser Met Ala Asn Ile Leu Ser Gly Ile Leu 30 35 40 gtt ttc gtc ctt gtcatc tgt gtt ctg gtt gct gtt atc cac gac caa 195 Val Phe Val Leu Val IleCys Val Leu Val Ala Val Ile His Asp Gln 45 50 55 tca caa caa aca atg gcgacc aac aac cat cag gga gaa gat aaa ccc 243 Ser Gln Gln Thr Met Ala ThrAsn Asn His Gln Gly Glu Asp Lys Pro 60 65 70 acc tcc gcc gcc acg ttc acagct ccg ttg cta caa gtt gat ctc aaa 291 Thr Ser Ala Ala Thr Phe Thr AlaPro Leu Leu Gln Val Asp Leu Lys 75 80 85 90 cgg gtt ccc gga aag ttg gaatcc aat gct gat gtt gag tgg caa cgc 339 Arg Val Pro Gly Lys Leu Glu SerAsn Ala Asp Val Glu Trp Gln Arg 95 100 105 tca gct tac cat ttt caa cccgat aag aat ttc atc agc gat cct gat 387 Ser Ala Tyr His Phe Gln Pro AspLys Asn Phe Ile Ser Asp Pro Asp 110 115 120 ggt cca atg tat cac atg gggtgg tac cat ctc ttc tac cag tac aac 435 Gly Pro Met Tyr His Met Gly TrpTyr His Leu Phe Tyr Gln Tyr Asn 125 130 135 cca gaa tca gcc ata tgg ggcaac atc aca tgg ggc cac tcc gta tca 483 Pro Glu Ser Ala Ile Trp Gly AsnIle Thr Trp Gly His Ser Val Ser 140 145 150 cga gac atg atc aac tgg ttccat ctc cca ttc gcc atg gtc ccg gac 531 Arg Asp Met Ile Asn Trp Phe HisLeu Pro Phe Ala Met Val Pro Asp 155 160 165 170 cat tgg tac gac atc gaaggg gtc atg acc gga tcc gcc acg gta ctc 579 His Trp Tyr Asp Ile Glu GlyVal Met Thr Gly Ser Ala Thr Val Leu 175 180 185 ccc aac ggt cag atc atcatg ctc tac act ggc aac gcg tac gat ctc 627 Pro Asn Gly Gln Ile Ile MetLeu Tyr Thr Gly Asn Ala Tyr Asp Leu 190 195 200 tcc cag tta cag tgc ttagca tac gcc gtc aac tca tct gat cct ctc 675 Ser Gln Leu Gln Cys Leu AlaTyr Ala Val Asn Ser Ser Asp Pro Leu 205 210 215 ctt ctg gaa tgg aaa aagtac gaa gga aac cca att ttg ttc cca ccg 723 Leu Leu Glu Trp Lys Lys TyrGlu Gly Asn Pro Ile Leu Phe Pro Pro 220 225 230 cct ggt gtg gga tac aaagat ttt cga gat cca tcc aca tta tgg atg 771 Pro Gly Val Gly Tyr Lys AspPhe Arg Asp Pro Ser Thr Leu Trp Met 235 240 245 250 ggt cct gat ggg gaatgg aga atg gta atg ggg tcc aaa cac aat gaa 819 Gly Pro Asp Gly Glu TrpArg Met Val Met Gly Ser Lys His Asn Glu 255 260 265 act att ggt tgt gcattg gtc tac cgt act act aat ttt acg cat ttt 867 Thr Ile Gly Cys Ala LeuVal Tyr Arg Thr Thr Asn Phe Thr His Phe 270 275 280 gaa ctg aat gag gaggta ctc cac gca gtc ccc cat act ggt atg tgg 915 Glu Leu Asn Glu Glu ValLeu His Ala Val Pro His Thr Gly Met Trp 285 290 295 gaa tgt gtg gac ctatac cct gtg tcc acc acg cac acg aat ggg ttg 963 Glu Cys Val Asp Leu TyrPro Val Ser Thr Thr His Thr Asn Gly Leu 300 305 310 gac atg aag gat aatggg ccg aat gtt aaa tat att ttg aaa caa agt 1011 Asp Met Lys Asp Asn GlyPro Asn Val Lys Tyr Ile Leu Lys Gln Ser 315 320 325 330 gga gac gaa gaccga cat gat tgg tat gcg gtt ggg act ttt gac cct 1059 Gly Asp Glu Asp ArgHis Asp Trp Tyr Ala Val Gly Thr Phe Asp Pro 335 340 345 gag aaa gat aagtgg tac cct gac gac cct gaa aac gat gtg gga atc 1107 Glu Lys Asp Lys TrpTyr Pro Asp Asp Pro Glu Asn Asp Val Gly Ile 350 355 360 ggg ttg aga tacgac tac gga aag ttc tat gcg tca aag aca ttt tat 1155 Gly Leu Arg Tyr AspTyr Gly Lys Phe Tyr Ala Ser Lys Thr Phe Tyr 365 370 375 gat caa cat gaaaag cgg agg gta ctt tgg ggt tat gtt ggt gaa acc 1203 Asp Gln His Glu LysArg Arg Val Leu Trp Gly Tyr Val Gly Glu Thr 380 385 390 gac ccc cct aagtcc gat ctt tta aag gga tgg gct aac atc ttg aat 1251 Asp Pro Pro Lys SerAsp Leu Leu Lys Gly Trp Ala Asn Ile Leu Asn 395 400 405 410 atc cca aggtcc gtt gtt ttg gac acg caa acc gga acc aat ttg att 1299 Ile Pro Arg SerVal Val Leu Asp Thr Gln Thr Gly Thr Asn Leu Ile 415 420 425 caa tgg ccgatt gat gaa gtg gaa aaa ttg aga tca aca aaa tat gac 1347 Gln Trp Pro IleAsp Glu Val Glu Lys Leu Arg Ser Thr Lys Tyr Asp 430 435 440 gaa ttc aaagac gtg gag ctc cga ccc gga tca ctc gtt ccc ctc gaa 1395 Glu Phe Lys AspVal Glu Leu Arg Pro Gly Ser Leu Val Pro Leu Glu 445 450 455 att ggc acagcg aca cag ttg gac ata agt gcg aca ttt gaa atc gat 1443 Ile Gly Thr AlaThr Gln Leu Asp Ile Ser Ala Thr Phe Glu Ile Asp 460 465 470 caa aag aagtta caa tca acg ctt gaa gcc gat gtt ttg ttc aac tgt 1491 Gln Lys Lys LeuGln Ser Thr Leu Glu Ala Asp Val Leu Phe Asn Cys 475 480 485 490 aca actagc gaa ggt tca gtc cgg aag ggt gtg ttg gga cca ttt gga 1539 Thr Thr SerGlu Gly Ser Val Arg Lys Gly Val Leu Gly Pro Phe Gly 495 500 505 atc gtggtt cta gcg gat gcc aac cgc tct gag caa ctt cct gtg tat 1587 Ile Val ValLeu Ala Asp Ala Asn Arg Ser Glu Gln Leu Pro Val Tyr 510 515 520 ttc tatatt gcc aaa gac acc gat gga acc tca aaa act tac ttc tgt 1635 Phe Tyr IleAla Lys Asp Thr Asp Gly Thr Ser Lys Thr Tyr Phe Cys 525 530 535 gct gatgaa tca agg tca tcg acg gac aaa tac gtt gga aaa tgg gta 1683 Ala Asp GluSer Arg Ser Ser Thr Asp Lys Tyr Val Gly Lys Trp Val 540 545 550 tac ggaagc agt gtt cct gtt ctt gaa ggt gaa aat tac aac atg agg 1731 Tyr Gly SerSer Val Pro Val Leu Glu Gly Glu Asn Tyr Asn Met Arg 555 560 565 570 ttactg gtg gat cat tcg ata gtg gaa ggg ttc gca caa gga gga aga 1779 Leu LeuVal Asp His Ser Ile Val Glu Gly Phe Ala Gln Gly Gly Arg 575 580 585 acggtg gtg aca tca aga gtg tac ccc acg aag gcc atc tat ggc gct 1827 Thr ValVal Thr Ser Arg Val Tyr Pro Thr Lys Ala Ile Tyr Gly Ala 590 595 600 gctaag ata ttt ttg ttc aac aac gcc acc gga att agc gtc aag gca 1875 Ala LysIle Phe Leu Phe Asn Asn Ala Thr Gly Ile Ser Val Lys Ala 605 610 615 tctctc aag atc tgg aaa atg gcg gaa gca caa ctc gat cca ttc cct 1923 Ser LeuLys Ile Trp Lys Met Ala Glu Ala Gln Leu Asp Pro Phe Pro 620 625 630 ctttct ggg tgg agt tct tgattattag aattcgtcat ccctctctat 1971 Leu Ser GlyTrp Ser Ser 635 640 ttgtgtgtta ttgttgtgaa atatggtagc atgattgcgggtttagtggg ggtattatgg 2031 tagtttgtta atggtggttg tggtactgca tttgtgagattataaattga attgttattc 2091 ctgtttacaa cttttctaag caaatggtat gtcatgttttgatcaaaaaa aaaaaaaaaa 2151 aaaaaa 2157 6 640 PRT Cichorium intybus 6 MetAla Ser Ser Thr Thr Ala Thr Thr Pro Leu Ile Leu Arg Asp Glu 1 5 10 15Thr Gln Ile Ser Pro Gln Leu Ala Gly Ser Pro Val Gly Arg Arg Leu 20 25 30Ser Met Ala Asn Ile Leu Ser Gly Ile Leu Val Phe Val Leu Val Ile 35 40 45Cys Val Leu Val Ala Val Ile His Asp Gln Ser Gln Gln Thr Met Ala 50 55 60Thr Asn Asn His Gln Gly Glu Asp Lys Pro Thr Ser Ala Ala Thr Phe 65 70 7580 Thr Ala Pro Leu Leu Gln Val Asp Leu Lys Arg Val Pro Gly Lys Leu 85 9095 Glu Ser Asn Ala Asp Val Glu Trp Gln Arg Ser Ala Tyr His Phe Gln 100105 110 Pro Asp Lys Asn Phe Ile Ser Asp Pro Asp Gly Pro Met Tyr His Met115 120 125 Gly Trp Tyr His Leu Phe Tyr Gln Tyr Asn Pro Glu Ser Ala IleTrp 130 135 140 Gly Asn Ile Thr Trp Gly His Ser Val Ser Arg Asp Met IleAsn Trp 145 150 155 160 Phe His Leu Pro Phe Ala Met Val Pro Asp His TrpTyr Asp Ile Glu 165 170 175 Gly Val Met Thr Gly Ser Ala Thr Val Leu ProAsn Gly Gln Ile Ile 180 185 190 Met Leu Tyr Thr Gly Asn Ala Tyr Asp LeuSer Gln Leu Gln Cys Leu 195 200 205 Ala Tyr Ala Val Asn Ser Ser Asp ProLeu Leu Leu Glu Trp Lys Lys 210 215 220 Tyr Glu Gly Asn Pro Ile Leu PhePro Pro Pro Gly Val Gly Tyr Lys 225 230 235 240 Asp Phe Arg Asp Pro SerThr Leu Trp Met Gly Pro Asp Gly Glu Trp 245 250 255 Arg Met Val Met GlySer Lys His Asn Glu Thr Ile Gly Cys Ala Leu 260 265 270 Val Tyr Arg ThrThr Asn Phe Thr His Phe Glu Leu Asn Glu Glu Val 275 280 285 Leu His AlaVal Pro His Thr Gly Met Trp Glu Cys Val Asp Leu Tyr 290 295 300 Pro ValSer Thr Thr His Thr Asn Gly Leu Asp Met Lys Asp Asn Gly 305 310 315 320Pro Asn Val Lys Tyr Ile Leu Lys Gln Ser Gly Asp Glu Asp Arg His 325 330335 Asp Trp Tyr Ala Val Gly Thr Phe Asp Pro Glu Lys Asp Lys Trp Tyr 340345 350 Pro Asp Asp Pro Glu Asn Asp Val Gly Ile Gly Leu Arg Tyr Asp Tyr355 360 365 Gly Lys Phe Tyr Ala Ser Lys Thr Phe Tyr Asp Gln His Glu LysArg 370 375 380 Arg Val Leu Trp Gly Tyr Val Gly Glu Thr Asp Pro Pro LysSer Asp 385 390 395 400 Leu Leu Lys Gly Trp Ala Asn Ile Leu Asn Ile ProArg Ser Val Val 405 410 415 Leu Asp Thr Gln Thr Gly Thr Asn Leu Ile GlnTrp Pro Ile Asp Glu 420 425 430 Val Glu Lys Leu Arg Ser Thr Lys Tyr AspGlu Phe Lys Asp Val Glu 435 440 445 Leu Arg Pro Gly Ser Leu Val Pro LeuGlu Ile Gly Thr Ala Thr Gln 450 455 460 Leu Asp Ile Ser Ala Thr Phe GluIle Asp Gln Lys Lys Leu Gln Ser 465 470 475 480 Thr Leu Glu Ala Asp ValLeu Phe Asn Cys Thr Thr Ser Glu Gly Ser 485 490 495 Val Arg Lys Gly ValLeu Gly Pro Phe Gly Ile Val Val Leu Ala Asp 500 505 510 Ala Asn Arg SerGlu Gln Leu Pro Val Tyr Phe Tyr Ile Ala Lys Asp 515 520 525 Thr Asp GlyThr Ser Lys Thr Tyr Phe Cys Ala Asp Glu Ser Arg Ser 530 535 540 Ser ThrAsp Lys Tyr Val Gly Lys Trp Val Tyr Gly Ser Ser Val Pro 545 550 555 560Val Leu Glu Gly Glu Asn Tyr Asn Met Arg Leu Leu Val Asp His Ser 565 570575 Ile Val Glu Gly Phe Ala Gln Gly Gly Arg Thr Val Val Thr Ser Arg 580585 590 Val Tyr Pro Thr Lys Ala Ile Tyr Gly Ala Ala Lys Ile Phe Leu Phe595 600 605 Asn Asn Ala Thr Gly Ile Ser Val Lys Ala Ser Leu Lys Ile TrpLys 610 615 620 Met Ala Glu Ala Gln Leu Asp Pro Phe Pro Leu Ser Gly TrpSer Ser 625 630 635 640

What is claimed is:
 1. An a33 1-SST enzyme encoding cDNA sequenceaccording to SEQ ID NO:
 1. 2. A recombinant DNA construct, recombinantgene or vector comprising the cDNA sequence according to SEQ ID NO: 1.3. A recombinant DNA construct, recombinant gene or vector comprisingmore than one copy of the cDNA sequence according to SEQ ID NO:
 1. 4. Amethod for producing a transgenic plant with a modified inulin producingprofile comprising in its genome a combination of a 1-SST(sucrose:sucrose 1-fructosyl-transferase) enzyme encoding gene and a1-FFT (fructan:fructan 1-fructosyl-transferase) enzyme encoding gene,said method comprising transforming an inulin producing host plantcomprising in its genome a combination of a 1-SST enzyme encoding geneand a 1-FFT enzyme encoding gene, by insertion of a recombinant genecontaining the 1-SST enzyme encoding a33 cDNA sequence according to SEQID NO: 1 into the genome of the host plant and wherein said 1-SST enzymeencoding gene, said 1-FFT enzyme encoding gene and said recombinant genecontaining the 1-SST enzyme encoding a33 cDNA sequence according to SEQID NO: 1, are respectively operably linked to a promoter sequence, aterminator sequence and optionally to a DNA sequence encoding atargeting signal or a transit peptide, all active in said host plant. 5.The method according to claim 4, wherein the recombinant gene comprisesmore than one copy of the cDNA sequence according to SEQ ID NO:
 1. 6. Amethod for producing a transgenic plant with a modified inulin producingprofile comprising in its genome a combination of a recombinant 1-SSTenzyme encoding gene comprising the 1-SST enzyme encoding a33 cDNAsequence according to SEQ ID NO: 1, and a recombinant 1-FFT enzymeencoding gene, said method comprising transforming a non-inulinproducing host plant by insertion into the genome of the host plant of arecombinant 1-SST enzyme encoding gene comprising the 1-SST enzymeencoding a33 cDNA sequence according to SEQ ID NO: 1 and of arecombinant 1-FFT enzyme encoding gene, wherein the recombinant 1-SSTenzyme encoding gene and the recombinant 1-FFT enzyme encoding gene arerespectively operably linked to a promoter sequence, a terminatorsequence and optionally a DNA sequence encoding a targeting signal or atransit peptide, all active in said host plant.
 7. The method accordingto claim 6, wherein the recombinant 1-SST enzyme encoding gene comprisesmore than one copy of the cDNA sequence according to SEQ ID NO:
 1. 8.The method according to claim 6 wherein the 1-FFT enzyme encoding DNAsequence of the recombinant 1-FFT enzyme encoding gene originates from aplant of the plant family of the Asteraceae (Compositae).
 9. The methodaccording to claim 6 wherein the recombinant 1-FFT enzyme encoding genecomprises at least one 1-FFT enzyme encoding c86b cDNA sequenceaccording to SEQ ID NO:
 3. 10. A method for producing a transgenic plantwith a modified inulin producing profile comprising in its genome arecombinant gene containing the a33 cDNA sequence according to SEQ IDNO: 1 coding for an enzyme which acts as a 1-SST enzyme as well as a1-FFT enzyme, said method comprising transforming a host plant which isfree of 1-FFT enzyme encoding genes by insertion into the genome of thehost plant of a recombinant gene containing the a33 cDNA sequenceaccording to SEQ ID NO: 1 which is operably linked to a promotersequence, a terminator sequence and optionally a DNA sequence encoding atargeting signal or a transit peptide, all active in said host plant.11. The method according to claim 10, wherein the host plant comprisesin its genome already a 1-SST enzyme encoding gene which is operablylinked to a promoter sequence, a terminator sequence and optionally aDNA sequence encoding a targeting signal or a transit peptide, allactive in said host plant.
 12. The method according to claim 4 whereinthe host plant is selected from the group consisting of wheat, barley,chicory, banana, and Jerusalem artichoke.
 13. The method according toclaim 6 wherein the host plant is selected from the group consisting ofcorn, rice, sorghum, millets, sunflower, cassava, canola, soybean, oilpalm, groundnut, cotton, sugar cane, bean, pea, cowpea, tomato, beet,sugar beet, tobacco, potato, sweet potato, coffee, cocoa and tea. 14.The method according to claim 10 wherein the host plant is selected fromthe group consisting of corn, rice, sorghum, millets, sunflower,cassava, canola, soybean, oil palm, groundnut, cotton, sugar cane, bean,pea, cowpea, tomato, beet, sugar beet, tobacco, potato, sweet potato,coffee, cocoa and tea.
 15. The method according to claim 4 whereininulin produced by the trangenic plant comprisesfructo-oligosaccharides.
 16. The method according to claim 6 wherein theinulin produced by the transgenic plant comprisesfructo-oligosaccharides.
 17. The method according to claim 10 whereinthe inulin produced by the transgenic plant comprisesfructo-oligosaccharides.
 18. The method according to claim 4 wherein theinulin produced by the transgenic plant has an average degree ofpolymerization of at least
 10. 19. The method according to claim 6wherein the inulin produced by the transgenic plant has an averagedegree of polymerization of at least
 10. 20. The method according toclaim 4 wherein said method comprises the subsequent steps of (i)preparing a recombinant 1-SST gene construct comprising the 1-SST enzymeencoding cDNA sequence according to SEQ ID NO: 1, operably linked to apromoter sequence, a terminator sequence, and optionally a DNA sequenceencoding a targeting signal or a transit peptide, all active in saidhost plant, (ii) inserting said recombinant 1-SST gene construct of stepi) into the genome of a cell of the host plant, and (iii) regenerating acorresponding transgenic plant from the transformed cell obtained instep ii).
 21. The method according to claim 6 wherein said methodcomprises the subsequent steps of (i) preparing a recombinant 1-SST geneconstruct comprising the 1-SST enzyme encoding cDNA sequence accordingto SEQ ID NO: 1, operably linked to a promoter sequence, a terminatorsequence, and optionally a DNA sequence encoding a targeting signal or atransit peptide, all active in said host plant, (ii) preparing arecombinant 1-FFT gene construct comprising a 1-FFT enzyme encoding DNAsequence from plant origin, operably linked to a promoter sequence, aterminator sequence, and optionally a DNA sequence encoding a targetingsignal or a transit peptide, all active in said host plant, (iii)inserting said 1-SST gene construct of step i) and said 1-FFT geneconstruct of step ii) into the genome of a cell of the host plant, and(iv) regenerating a corresponding transgenic plant from the transformedcell obtained in step iii).
 22. The method according to claim 21 whereinthe recombinant 1-FFT gene construct comprises at least one 1-FFT enzymeencoding c86b cDNA sequence according to SEQ ID NO:
 3. 23. The methodaccording to claim 10 wherein said method comprises the subsequent stepsof (i) preparing a recombinant gene construct comprising the enzymeencoding cDNA according to SEQ ID NO: 1, operably linked to a promotersequence, a terminator sequence, and optionally a DNA sequence encodinga targeting signal or a transit peptide, all active in said host plant,(ii) inserting said recombinant gene construct of step i) into thegenome of a cell of the host plant, and (iii) regenerating acorresponding transgenic plant from the transformed cell obtained instep ii).
 24. The method for producing inulin with a modified inulinprofile from source plant material wherein the source material is plantmaterial from a transgenic plant obtained by the method defined in claim4.
 25. A method for producing inulin with a modified inulin profile fromsource plant material wherein the source material is plant material froma transgenic plant obtained by the method defined in claim
 6. 26. Themethod for producing inulin with a modified inulin profile from sourceplant material wherein the source material is plant material from atransgenic plant obtained by the method defined in claim
 10. 27. Arecombinant gene or vector each comprising a combination of a 1-SSTenzyme encoding gene comprising the cDNA sequence according to SEQ IDNO: 1, and of a 1-FFT enzyme encoding gene of plant origin, which enzymeencoding genes are respectively operably linked to a promoter sequenceand a terminator sequence, and optionally to a DNA sequence encoding atargeting signal or a transit peptide, all active in an host plant. 28.The recombinant gene or vector according to claim 27, wherein the 1-FFTenzyme encoding gene comprises at least one cDNA sequence according toSEQ ID NO:
 3. 29. A transgenic plant with a modified inulin producingprofile; or a root, shoot, plant part, plant tissue, plant cell thereofor seed thereof, each with a genome according to that of said transgenicplant; comprising in its genome a recombinant 1-SST enzyme encoding genecomprising at least one copy of the cDNA sequence according to SEQ IDNO: 1, operably linked to a promoter sequence and a terminator sequence.30. A transgenic plant with a modified inulin producing profile; or aroot, shoot, plant part, plant tissue, plant cell thereof, each or seedthereof with a genome according to that of said transgenic plant,according to claim 29; comprising in its genome a recombinant 1-SSTenzyme encoding gene comprising more than one copy of the cDNA sequenceaccording to SEQ ID NO: 1, operably linked to a promoter sequence and aterminator sequence.
 31. A transgenic plant with a modified inulinproducing profile; or a root, shoot, plant part, plant tissue, plantcell thereof or seed thereof, each with a genome according to that ofsaid transgenic plant, according to claim 29; comprising in its genomefurthermore a 1-FFT enzyme encoding gene operably linked to a promotersequence and a terminator sequence.
 32. A transgenic plant with amodified inulin producing profile; or a root, shoot, plant part, planttissue, plant cell thereof or seed thereof, each with a genome accordingto that of said transgenic plant, according to claim 30, comprising inits genome furthermore a 1-FFT enzyme encoding gene operably linked to apromoter sequence and a terminator sequence.
 33. A transgenic plant witha modified inulin producing profile; or a root, shoot, plant part, planttissue, plant cell thereof or seed thereof, each with a genome accordingto that of said transgenic plant, according to claim 31; comprising inits genome a 1-FFT enzyme encoding gene that comprises at least one copyof the cDNA sequence according to SEQ ID NO:
 3. 34. A transgenic plantaccording to claim 29, which presents an improved tolerance againstdrought stress or cold stress.
 35. A transgenic plant according to claim30, which presents an improved tolerance against drought stress or coldstress.
 36. A transgenic plant according to claim 31, which presents animproved tolerance against drought stress or cold stress.
 37. Atransgenic plant according to claim 32, which presents an improvedtolerance against drought stress or cold stress.
 38. A transgenic plantaccording to claim 33, which presents an improved tolerance againstdrought stress or cold stress.